Study the Relationship Between Apoptosis Activity ...

REPUBLIC OF IRAQ Ministry Of Higher Education and Scientific Research University Of Baghdad Genetic Engineering and Biotechnology Institute for Postgraduate Studies

University of Baghdad

Study the Relationship Between Apoptosis Activity and Mutations of the Gene bcl2 in companied with Resistance to Chemotherapy in Chronic Lymphocytic Leukemia Patients. A Thesis Submitted to the Council of the Genetic Engineering and Biotechnology Institute for Postgraduate Studies, University of Baghdad, In Partial Fulfillment of the Requirements for the Master degree in Genetic Engineering and Biotechnology By Manal Lafta Abdul-Hassan B.Sc. Veterinary, Univ. of Baghdad 1999

Supervised by Prof. Dr. Abdul-Hussein M. Al-Faisal August 2013

Ramadan 1434 1

Summary This study investigates the relationship between apoptosis activity, mutations of the gene bcl2 accompanying with resistance to chemotherapy in leukemic patients. The study includes 30 Iraqi patients with chronic lymphocytic leukemia (CLL) and 15 healthy people . Blood samples were collected from the center of hematology /Yarmouk hospital from September 2012 till January 2013 .They were clinically diagnosed by the consultant medical staff of the center. Apoptosis detection study was carried out to detect intracellular bcl-2 ,using ficoll solution (lymphoprep) to isolate lymphocytes which were incubated with annexine V-FITC kit to detect the apoptosis activity. The results showed that the necrotic cells percent was found to be significantly 1T

higher in the patients´ samples(1.07 ± 0.03) than healthy (0.17 ± 0.02). 1T

1T

1T

1T

1T

Apoptosis cell percent expression was found to be significantly higher in the 1T

patients´ samples (15.04 ± 1.03) than healthy (4.40 ± 0.5) with a range of 1T

1T

1T

1T

(1.1 - 41.2) Late apoptosis percent expression was 0.639 % in patients and 1T

0.803 percent in healthy group. No association was found between annexin-V expression and the age 1T

and gender of patients. The molecular study involved DNA extraction by using Qiagene kit, DNA electrophoresis through the gel and polymerase chaine reaction(PCR) for bcl2 gene ,exon 1 and sequencing of 10 CLL samples ,(with high apoptosis activity) showed that 78 mutations were observed in bcl2 gene. 34 of them were in P1(43.58%) and 44 of them were in P2(56.41%). (Substitution= 34.61% P1 and 39.74% P2) and (deletion =8.97% P1 and 16.66% P2). Most of CLL samples showed compound mutations which 2

may involve multiple deletions and substitutions in the same site that leads to defect protein. And presence of multiple frequency in the same site and type. The results of the effect of the mutations was found (Missense 20.51% in P1 and 33.33% P2) ,(Silent 10.25% P1 and 1.28% P2) ,(Frameshift 8.97% P1 and 16.66% P2) and (Nonsense 2.56% P1 and 1.28% P2).

Table of Contents Summary ……………………………………………………………… I List of Contents ………………………………………………………..II I List of Tables ……………………………………………………......... V 3

I List of Figures ……………………………………………………….. V II List of Abbreviations ……………………………………………..... V II I

Chapter One: Introduction 1-1 Introduction ……………………………………………..….……….1 1-2 The aim of this study………………………………………………..2

Chapter Two: Literature Review 2-1 Leukemia……………………………………….………...…,.............3 2-1.1 Classification……………………………....…..………………. ...3 2-1.2 History of leukemia.……………….………..…………………… ..6 2-1-3 Causes…………………….. ….…………………..…………….…7 2-1-4 Diagnosis………………...………………………………………9 2-2 Chronic Lymphocytic Leukemia…………………………….……10 2-2-1 Staging of Chronic Lymphocytic Leukemia..…………………..10 2-2.2 CLL development………….………….……………...…..………12 2-2.3 CLL and Immune System..………………….……………..…..12 2-3 Apoptosis………………………...………..……….……………….14 2-3.1 History of Apoptosis..…….… ………………………………….15 2-3-2 bcl2 and Apoptosis…....……………………………………….15 2- 3.2.1 Apoptosis regulater bcl2…….……………………………..15 2-32.2 Function………….………………………...........…………….17 2-3.3 Apoptotic Mechanism..……..……..................………………...18 2-3-3.1 Extrinsic Pathway of Apoptosis...……………...…………….20 2-3.3 .2 Intrinsic Pathway of Apoptosis…….…..……………..……….20 2-4 Flow cytometry…………….. ….………………….………...........24

4

2.5 Chemotherapy………………………………………………………25 2-5.1 Alkylating Agents…………. ………...………… ………..….....26 2.5.2 Purine Nucleoside analogs………………………………………27

Chapter Three: Materials & Methods 3-1 Materials ……………………………………………………….…29 3-1-1 Laboratory Equipments……... ……………………………..…….29 3-1-2 Chemical Materials…………………………......…….........…….30 3-2 Methods……………………………………...….……….................31 3-2.1 Specimens Collection...…………………………..…….……...31 3-2-1.1 Patients Selection ….………………………..………….…......31 3-2-1.2 Blood Sampling………………………………………….…….31 3-2-2 Isolation of Lymphocytes.……. ……………….………….…… 31 3-2-2-1.preparation of Solutions………….……………………………..31 3-2.2.2 Isolation Protocol of Lymphocytes……..…………………….32 3-2-3Detection of Apoptosis in the Isolated Lymphocytes………....33 3-2-3.1 Principle…………………………..…………………….….......33 3-2-3.2 Procedure……………………… …………..….…....................34 3-2-4 DNA Extraction………………………………………………....35 3-2-4.1 Kit components…………………….……………. …………..35 3-2-4.2 Procedure of DNA Extraction……………….……...…….….35 3-2-5 DNA Checking…….……………………………………………..36 3-2-6 Agarose Gel Electrophoresis... …………………………………..37 3-2-6-1 Protocol……………………........................................................37 3-2-7 Polymerase Chain Reaction PCR ….……………………………39 3.2.7.1 PCR Working Solutions…………………………………..…..40 3.2.7.2 PCR Protocol…………………………………………………41 3.2.7.2 PCR Products Sequencing…..………....…................................43

5

3.8 Statical Analysis………………………………………………….…44

Chapter foure: Results & Discussion 4.1 Subject data ………………….……………………………………45 4.1.1 Age …………………………………………………………….…45 4.2 Flow Cytometry Annexin V-FITC Apoptosis Detecion…………..45 4.3 Molecular Analysis of bcl 2 Gene…………………….……………54 4.3.1 Extraction of Blood DNA………………………………………54 4.3.2 bcl 2 PCR Analysis………………………………………………55 4.3.3 Detection of bcl2 Mutations in CLL Patients ………………….....57 4.3.3.1 Percentage of Mutations Types….…………...…………………64 4.3.3.2 Effect of Mutations……………………………………………..65 4.3.3.3 Compound Mutations..…………..……………………………..68 4.3.3.4 Frequency of Mutations………………………………………68

List of Tables No.

Tables

Page

3.1

General laboratory equipments

31

3.2

Chemical materials

32

3.3

Primer sequence for amplification bcl2 gene

39

6

3.4

Master mix components of bcl2 PCR (P1)

40

3.5

Master mix components of bcl2 PCR (P2)

41

3.6

The PCR program for P1 region of bcl2 gene amplification

42

3.7

The PCR program for P2 region of bcl2 gene amplification

42

3.8

ProFi Taq PCR premix components of bcl2 gene( P1&P2)

43

3.9

The ProFi Taq PCR premix condition for P1& P2 regions of bcl2 44 gene amplification

4.1

Score range and apoptosis % in lymphocyte isolated from CLL

48

patients by flow cytometry machine 4.2

Score range and apoptosis % in lymphocyte isolated from healthy

49

(control) by flow cytometry machine . 4.3

Flow cytometer measurements of patients& control

50

4.4

Analysis of bcl2 gene (p1) in CLL patients group

60

4.5

Analysis of bcl2 gene (p2) in CLL patients group

63

4.6

Percentage of mutations type in CLL patients group

64

4.7

Percentage of mutation effect in bcl2 gene in CLL Patients

66

4.8

Number of mutations repeated in CLL patients

69

List of figures No. 2.1

Figures bcl2 family

Page 17

2.2

Mechanism of apoptosis

19

2.3

Morphological changes of apoptotic cells

22

3.1

The isolation of lymphocyte layer

33

3.2

Schematic diagram of the bcl2 gene &the PCR primers used

39

7

4.1

Annexine V expression for apoptosis analysis for control samples

46

4.2

Annexine V expression for apoptosis analysis for CLL &healthy

47

Samples

4.3 4.4

The chromosomal DNA electrophorsed on Agarose gel (1%) ,70 volt/15min. PCR product electrophoresed on 1.5% agarose gel 70 volt/hr. P1

54 55

produc (290 bp)/P2 product (367bp)/Negative sample as control

4.5

PCR product electrophoresed on 1.5% agarose gel 70 volt/hr. P1

56

product (290 bp)/P2 product (367bp)/Negative sample as control.

4.6

Sequencing analysis of bcl2 gene primer 1(393-411site)

58

4.7

. Sequencing analysis of bcl2 gene primer 1(515site)

59

4.8

Sequencing analysis of bcl2 gene primer 2 (1149 site)

61

4.9

Sequencing analysis of bcl2 gene primer 2 (822-842 site)

62

List of Abbreviations AIF

Apoptosis inducing factor

ALL

Acute Lymphoid Leukemia

AML

Acute Myeloid Leukemia

Apaf-1

Apoptotic protease activating factor

Apo -1 L

Apoptosis 1 ligand

B-Cells

Bone marrow cells

BAD

BCL2 antagonist of cell death 8

BAG

BCL2 associated athano gene

BAK

BCL2 antagonist killer 1

BAX

BCL2 associated X protein

Bcl-w

BCL2 like 2 protein

Bcl-x

BCL2 like 1

Bcl-XL

BCL2 related protein, long isoform

BH1 toBH4

Bcl-2 homology domains

BID

BH3 interacting domain death agonist

BIK

BCL2 interacting killer

BIM

BCL2 interacting protein

Caspase-8

Cysteinyl aspartic acid protease8

CLL

Chronic Lymphocytic Leukemia

CML

Chronic Myelogenous Leukemia

CR

Complete Response

CT scans

Computed Tomography

DEDs

Death effector domains

DISC

death inducing signaling pathway complex

DLCL

diffuse large cell lymphoma

EDTA

Diethylene diamine tetra acetic acid

ELF

Electromagnetic Low Frequency

Endo G

Endonuclease G

ER FADD

Endoplasmic Reticulum Fas Associated Death Domain

Fas/L

Fatty Acid Synthetase Ligand

FCM

Flowcytrometric Analysis

FITC

Fluorescein Isothiocyanate

FSC

Forward Scatter

9

HCL

Hairy Cell Leukemia

HIV

Human Immunodificiency Virus

HTLV

Human T-Lymphotropic Virus

IAP

Inhibitor of Apoptosis Proteins

Ig

Immunoglobulin

MAC

mitochondrial apoptosis induced channel

MHC

Major histo component complex

MOMP

mitochondrial outer membrane permeabilization

MRI

Magnetic Resonance Imaging

NHL

Non-Hodgkin lymphoma

NK cells

Natural killer cells

OS

overall survival

PBLs

Perepheral Blood Lymphocytes

PCR

Polymerase Chain Reaction

PI

Propidium iodide

PS

phosphatidylserine

RBCs

Red blood cells

SLL

small lymphocytic lymphoma

Smac/DIABLO

Second mitochondrial activator of caspases /direct IAp binding protein with low PI

SNP

single nucleotide polymorphism

SSC

Side Scatter

T cells

Thymus Cells

TC

Cytotoxic T

TCR

T Cell Receptor

TH

T helper

TM

trans membrane domain

TNF

Tumor necrosis factor 10

T-PLL

T-cell prolymphocytic leukemia

Treg

T regulatory

UV

Ultra violet

WBCs

White Blood Cells

WHO

World Health Organization

11

Chapter One

Introduction

1.1 : Introduction Leukemia is defined as the

accumulations of single, hematopoietic

cells in the bloodstream or lymph (Rai et al., 1975).Generic cells (hematopoietic stem cells) mature into either myeloid or lymphoid ancestral cells from which all other specialized cells arise (Angelica,2011). Leukemias occur when there is a malfunction in the lymphoid lineage from which natural killer cells, T cells, B cells and dendritic cells are derived. CLL lymphocytes develop from normal B lymphocytes (Chiorazzi et al., 2005). When CLL is present, mutated lymphocytes that cannot perform their disease-fighting role reproduce uncontrollably, they populate the bone marrow then build up in the bloodstream, hindering the production of other cells. There is no cure for CLL (Hamblin, 2001). Generally, CLL is treated with chemotherapy which has side effects that can be as devastating as the disease itself. Apoptosis, the cell-suicide programme executed by caspases, is critical for maintaining tissue homeostasis, and impaired apoptosis is now recognized to be a key step in tumorigenesis (Rajesh et al., 2009). Whether a cell should live or die is largely determined by the bcl-2 family of anti- and proapoptotic regulators. These proteins respond to cues from various forms of intracellular stress, such as DNA damage or cytokine deprivation, and interact with opposing family members to determine whether or not the caspase proteolytic cascade should be unleashed (Rajesh et al., 2009). Iraq 12

statistics and epidemiological studies have shown that leukemia is the most common malignancies in males and in females and the major form of pediatric cancer (WHO, 2008).

Chapter One

Introduction

Chemotherapy is a leukemia treatment that uses drugs to stop cell division (National cancer institute, 2007). There are different types of chemotherapy, each with its own target and goal. Some aim to wipe out the cancer while others try to stop cancer from progressing while treating any symptoms (Rai et al., 2005). Many strategies are used to enhance cell death one of them is targeting bcl2, or engaging the death receptor pathway. The most important problem of the treatment is the drug resistance which makes the treatment difficult.The number of patients with leukemia have increased in Iraq dramatically following the first Gulf War ,this might be attributed to exposure to depleted uranium (a radioactive element used in ammunition) as well as to exposure to other toxic environmental pollutants (Essa et al. ,2007).

1.2 Aims of study The present study aims to investigate : 1-The relationship between apoptosis activity and mutations in the gene bcl2. 2-Chemotherapy resistance in lymphocytic leukemia patients. 3-Detection of the mutations in bcl2 by sequencing .

13

Review of Literature

Chapter Two

2.1 Leukemia Leukemia (American English) or leukaemia (British English) is a type of cancer of the blood or bone marrow characterized by an abnormal increase of immature white blood cells called blasts (Rai et al., 1975). Leu Leukemia is a broad term covering a spectrum of diseases. In turn, it is part of the even broader group of diseases affecting the blood, bone marrow and lymphoid system (Eyal and Attar,2010).

2.1.1 Classific 1T

0T1

0T29

ation Clinically and pathologically, leukemia is subdivided into a variety of 1T

large groups. The first division is between its acute and chronic forms. 0T1

0T1

0T1

0T1

The four major kinds of leukemia: Acute leukemia is characterized by a rapid increase in the number of 24T

24T0

0T1

immature blood cells. Crowding due to such cells makes the bone marrow unable to produce healthy blood cells. Immediate treatment is required in acute leukemia due to the rapid progression and accumulation of the malignant cells, which then spill over into the bloodstream and spread to other organs of the body. Acute forms of leukemia are the most common forms of leukemia in children (Jameson et al., 2005). 0T1

0T

Chronic leukemia is characterized by the excessive build up of relatively 0T

0T1

mature, but still abnormal white blood cells. Typically taking months or

14

years to progress, the cells are produced at a much higher rate than normal, resulting in many abnormal white blood cells (Rai et al., 1975).


Chapter Two

Chronic leukemia mostly occurs in older people, but can theoretically occur in any age group (Binet et al.,1981). Additionally, the diseases are subdivided according to which the kind of blood cell is affected. This split divides leukemias into lymphoblastic or lymphocytic leukemias and myeloid or

myelogenous leukemias

(Jameson et al.,2005). • In lymphoblastic or lymphocytic leukemias, the cancerous change takes place in a type of marrow cell that normally goes on to form lymphocytes which are infection-fighting immune system cells. Most lymphocytic leukemias involve a specific subtype of lymphocyte, the B cell. • In myeloid or myelogenous leukemias, the cancerous change takes place in a type of marrow cell that normally goes on to form red blood cell, some other types of white cells, and platelets. Combining these two classifications provides a total of four main categories. 1T

•

Acute lymphoblastic leukemia (ALL) is the most common type of leukemia in young children. This disease also affects adults, especially those

between

65 years and older. Standard treatments

involve chemotherapy and radiotherapy. The survival rates vary by 15

age: 85% in children and 50% in adults (Jameson et al., 2005) .Subtypes include precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitts leukemia, and acute biphenotypic leukemia.

Chapter Two •


Chronic lymphocytic leukemia (CLL) most often affects adults over the age of 55years.It sometimes occurs in younger adults, but it almost never affects children. Two-thirds of affected people are men. The five-year survival rate is 75% (Rai et al., 1975).. It is incurable, but there are many effective treatments. One subtype is B-cell pro lymphocytic leukemia, a more aggressive disease (Binet et al.,1981).

•

Acute myelogenous leukemia (AML) occurs more commonly in adults than in children, and more commonly in men than women. AML is treated with chemotherapy. The five-year survival rate is 40% (Colvin and Elfenbein, 2003).

•

Chronic myelogenous leukemia (CML) occurs mainly in adults; a very small number of children also develop this disease. One subtype is the chronic monocytic leukemia (Colvin and Elfenbein, 2003).

•

Hairy cell leukemia (HCL) is sometimes considered a subset of chronic lymphocytic leukemia, but does not fit neatly into this pattern. About 80% of affected people are adult men. HCL is incurable, but easily treatable. Survival is 96% to 100% at ten years

( Else et

al.,2005) . •

T-cell prolymphocytic leukemia (T-PLL) is a very rare and aggressive leukemia affecting adults; somewhat more men than women are diagnosed with this disease ( Matutes ,1998). Despite its 16

overall rarity, it is also the most common type of mature Tcell leukemia (Valbuena et al., 2005). Nearly all other leukemias involve B cells. It is difficult to treat, and the median survival is measured in months.

Chapter Two •


Large granular lymphocytic leukemia may involve either T-cells or NK cells ; like hairy cell leukemia, which involves B cells (Elaine et al.,2001) .

•

Adult T-cell leukemia is caused by human T-lymphotropic virus (HTLV), a virus similar to HIV. Like HIV, HTLV infects CD4+ Tcells and replicates within them. (Valbuena et al., 2005).

2.1.2 History of leukemia Leukemia was first observed by pathologist Rudolf Virchow in 1845, observing an abnormally large number of white blood cells in a blood sample from a patient, Virchow called the condition Leukämie in German, which he formed from the two Greek words( leukos ), meaning "white", and( aima ), meaning "blood"(Patlak,2010). Around ten years after Virchow's findings, pathologist Franz Ernst Christian Neumann found that one deceased leukemia patient's bone marrow was colored "dirty greenyellow" as opposed to the normal red, this finding allowed Neumann to conclude that a bone marrow problem was responsible for the abnormal blood of leukemia patients(Eyal and .Attar,2010). By 1900 leukemia was viewed as a family of diseases as opposed to a single disease. In 1962, researchers Emil J. Freireich Jr. and Emil Frei III 17

used combination chemotherapy in an attempt to cure leukemia. The tests were successful with some patients surviving long after the tests (Patlak , 2010) .

Chapter Two


2.1.3 Causes No single known cause for any of the different types of leukemia exists. The known causes, which are not generally factors within the control of the average person, account for relatively few cases (Ross et al., 2002). The different leukemias are likely to have different causes. • Leukemia, like other cancers, results from mutations in the DNA. Certain mutations can trigger leukemia by activating

oncogenes or

deactivating tumor suppressor genes like P53, and thereby disrupting the regulation of cell death, differentiation or division. These mutations may occur spontaneously or as a result of exposure to radiation or carcinogenic substances (WHO,2002). • Among adults, the known causes are natural and artificial ionizing radiation, a few viruses such as human T-lymphotropic virus , and some chemicals, notably benzene (Stass et al.,2000 ; Robinette et al. ,2001;Wiernik , 2001). The use of tobacco is associated with a small increase in the risk of developing acute myeloid leukemia in adults (Wiernik , 2001). Cohort and case-control studies have linked exposure to some petrochemicals and hair dyes to the development of some

forms

of

leukemia.

A

few

cases

of

maternal-fetal

transmission have been reported (Koren and Lishner ,2010).

18

•

Diet has very limited or no effect, although eating more vegetables may confer a small protective benefit (Ross et al. , 2002).

• Viruses have also been linked to some forms of leukemia. Experiments on mice and other mammals have demonstrated the relevance of retroviruses in leukemia, and human retroviruses have also been identified, the first human retrovirus identified was human Chapter Two


T-lymphotropic virus, or HTLV-1, which is known to cause adult T-cell leukemia ( Leonard, 1998). • Some people have a genetic predisposition towards developing leukemia. This predisposition is demonstrated by family histories and twin studies. The affected people may have a single gene or multiple genes in common. In some cases, families tend to develop the same kinds of leukemia as other members; in other families, affected people may develop different forms of leukemia or related blood cancers (Elaine et al. , 2001) .In addition to these genetic issues, people with chromosomal abnormalities or certain other genetic conditions have a greater risk of leukemia for example, people with Down syndrom have a significantly increased risk of developing forms of acute leukemia (especially acute myeloid leukemia) (Robinette et al.,2001). • Whether non-ionizing radiation causes leukemia has been studied for several

decades.

The International

Agency

for

Research

on

Cancer expert working group undertook a detailed review of all data on static and extremely ELF( Electromagnetic Low Frequency) energy, which occurs naturally and in association with the generation, transmission, and use of electrical power (WHO,2002) , they concluded that there is limited evidence that high levels of ELF magnetic (but not 19

electric) fields might cause childhood leukemia. Exposure to significant ELF magnetic fields might result in two fold excess risk for leukemia for children exposed to these high levels of magnetic fields. • A study conducted at the center for research in epidemiology and population health in France showed that children born to mothers who use fertility drugs to induce ovulation are more than twice as likely to Chapter Two


develop leukemia during their childhoods than other children (Rudant et al. , 2012).

2.1.4 Diagnosis Diagnosis is usually based on repeated complete blood counts and a bone marrow examination following observations of the symptoms, however, in rare cases blood tests may not show if a patient has leukemia, this is usually because the leukemia is in the early stages or has entered remission(Abbott,2005). A lymph node biopsy can be performed as well in order to diagnose certain

types

of

leukemia

in

certain

situations

(Eyal

and

Attar,2010). Following diagnosis, blood chemistry tests can be used to determine the degree of liver and kidney damage and the effects of chemotherapy on the patient like reticulocytes count, examination of the peripheral blood smear, iron studies including erythropoietin level and

Fe(Ferous) and ferritin,

bone marrow biopsy with cytogenetics. When

concerns arise about visible damage due to leukemia, doctors may use an Xray, MRI, or ultrasound , these can potentially view leukemia's effects on such body parts as bones (X-ray), the brain (MRI), or the kidneys, spleen,

20

and liver (ultrasound). Finally, CT scans are rarely used to check lymph nodes in the chest (Eyal and .Attar,2010). Despite the use of these methods to diagnose whether or not a patient has leukemia, many people have not been diagnosed because many of the symptoms are vague , and can refer to other diseases.

Chapter Two


2.2 Chronic Lymphocytic Leukemia Chronic lymphocytic leukemia, often referred to as CLL, is a type of cancer of the blood. It is one of a group of diseases that affects a type of white blood cell called a lymphocyte. The lymphocytes help the body fight infection.CLL and small lymphocytic lymphoma "SLL" are variants of the same disease. In chronic lymphocytic leukemia, abnormally high numbers of ineffective lymphocytes are found in the blood and/or bone marrow (the spongy area in the middle of large bones where blood cells are formed) (Kindt et al., 2007).In SLL, these same cells are commonly found in the lymph nodes. The abnormal cells cannot fight infection as normal lymphocytes do, but instead collect in lymph nodes and other areas, such as the liver and spleen. The accumulation of ineffective lymphocytes can interfere with the production of other blood cells, such as red cells and platelets (Chiorazzi et al.,2005) .Unlike other types of leukemia, CLL usually progresses slowly. In many cases, it causes the patient little, if any, problems in its early stages. Patients may live with CLL for decades. Some patients, however, live for a shorter period. Many times, it is diagnosed, only incidentally, by blood tests that are performed during a routine physical exam (Abbott, 2005). 21

2.2.1 Staging of chronic lymphocytic leukemia The natural history of CLL may vary considerably from one person to another. Some people become sick within a short time of diagnosis; others live comfortably for years without problems. Determining which patients are most likely to get sick, and therefore most likely to benefit from treatment, is an important task (Rai et al. ,1975).

Chapter Two


Two systems for staging CLL are now in use. Both systems are based upon results of the physical examination and blood tests. The Rai system is based on an analysis of how the body is affected by the abnormal lymphocytes (Rai et al.,1975). There are five stages. The higher numbers indicate a more advanced stage of disease:  Stage 0: Increased numbers of abnormal lymphocytes are found in the blood or bone marrow; lymph nodes/organs are not swollen; and production of red cells and platelets is normal  Stage I: Increased abnormal lymphocytes and enlarged lymph nodes  Stage II: Increased abnormal lymphocytes with enlarged liver or spleen, with or without enlarged lymph nodes  Stage III: Increased abnormal lymphocytes with anemia (low red blood cell count), with or without an enlarged spleen, liver, or lymph nodes  Stage IV: Increased abnormal lymphocytes with a low platelet count, with or without anemia, enlarged liver, spleen, or lymph nodes. The Binet system This system considers the five possible sites where lymphocytes can collect (lymph nodes in the neck, armpit, and groin;

22

lymphocyte-containing channels in the spleen and liver, and also whether anemia or low platelet counts are present) ( Binet et al .,1981) . There are three stages: Stage A: Fewer than three involved sites  Stage B: Three or more involved sites  Stage C: Presence of anemia or low platelet counts Staging CLL helps determine how likely it is that the patients will Chapter Two


develop serious problems related to their illness. Patients at Rai stage 0 are considered at low risk, those at stages I or II are at intermediate risk, and those at stage III or IV are at high risk. Similarly, patients characterized according to the Binet system have progressively increasing risk, with stage A as the lowest and stage C as the highest risk group (Binet et al .,1981).

2.2.2 CLL development It is a leukemia that originates in the bone marrow with the lymphoid line of cell development. Unlike cancers typified by tissue tumors (carcinomas, sarcomas, lymphomas), leukemias are accumulations of single, hematopoietic cells in the bloodstream or lymph (Kindt et al., 2007). …Generic cells (hematopoietic stem cells) mature into either myeloid or lymphoid ancestral cells from which all other specialized cells arise. …Leukemias occur when there is a malfunction in the lymphoid lineage from which natural killer cells, T cells, B cells and dendritic cells are derived. CLL lymphocytes develop from normal B lymphocytes (Chiorazzi et al., 2005). When CLL is present, mutated lymphocytes cannot perform their disease-fighting role uncontrollably. They are in intermediate stage of B-

23

cell cycle between pre-B and mature B-cell (Eyal and .Attar,2010). They populate in the bone marrow then build up in the bloodstream, hindering the production of other cells.

2.2.3 CLL and immune system There are two arms to the human immune system: innate (natural, inherited and nonspecific) and adaptive responses (Kindt et al., 2007). CLL is thought to have its origins in the adaptive component of the immune system which consists of a highly specific detection and removal of foreign invaders (Chiorazzi et al., 2005). Review of Literature

Chapter Two

Adaptive immunity is characterized by an ability to recognize and remember these foreign antigens .The adaptive immune system also discriminates between foreign and body cells and between varieties of antigens. The main agents of adaptive immunity are white blood cell lymphocytes. Normally, generic stem cells propagate along the lymphoid line of development to become one of three types of specialized lymphocytes, first natural killer (NK) cells are nonspecific immune cells that belong to the natural innate immune system (Kindt et al., 2007; Coleman, 2010), the other two types of lymphocytes in the lymphoid lineage are T-cells and Bcells; these

are part of the highly specific adaptive immune system, with

cell surface proteins that enable antigen-specific recognition and response . … T cells recognize specific antigens only when their matching antigen is attached to another cell’s MHC (cell-surface proteins that present antigen). Hh There are many types of T cells which become either active or memory T cells upon encountering their antigen in conjunction with an MHC (Kindt et al., 2007; Coleman, 2010). In activated form, T helper (Th) secrete cytokine signalling proteins to induce response of other immune agents or 24

function as long-lasting memory cells. Upon antigen exposure, cytotoxic T (Tc) cells become memory cells or cytotoxic T lymphocytes .T regulatory (Treg) cells down-regulate the immune system when activated, playing a repressive role. The other type of lymphocyte, B cells, the site of B cell origination and maturation is the bone marrow and they produce proteins specific to a certain foreign antigen and present these antibody proteins on their cell surface (Chiorazzi et al. , 2005). B cells are the lymphocytes that are central to CLL. Chapter Two


Here, genetic rearrangement takes place, yielding B cells with different membrane antibodies that function as antigen receptors. Encounters between these mature B cells and their antigens take place within the lymph system (Nester et al., 2007; Coleman, 2010). Fluid full of mature lymphocytes (lymph) circulates through lymphatic vessels, tissues, organs like the spleen, and lymph nodes. Lymph nodes act as filters, bringing invading antigens or malfunctioning host cells and B cells in contact in order for binding to stimulate B cell activation and consequent immune response. CLL is thought to develop from antigen activated B cells (Chiorazzi et al., 2005). If a T helper (Th) receptor recognizes the antigen, it will signal the presenting B cell to proceed with production of a population of clone B cells (proliferation). During this reproduction, DNA undergoes genetic rearrangements that affect the antigen-binding of the clones’ antibodies (Kindt et a l., 2007).

25

2.3 Apoptosis Apoptosis has been recognized as a typical and important mode of programmed cell death, which involves the genetically determined elimination of cells (Guicciardi and Gores, 2009). It is the process of programmed cell death (PCD) that may occur in multi cellular organisms (Green , 2011). Biochemical events lead to characteristic cell changes (morphology) and death. In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis, in general, confers advantages during an organism's life cycle, for example, the differentiation of fingers and toes

in a developing human embryo occurs in, cells between the

fingers apoptose (Reed and Tomaselli, 2000; Elmore, 2007) . Chapter Two


The result is that the digits are separate. Unlike necrosis, apoptosis produces cell fragments called apoptotic bodies that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells, and cause damage (Alberts et al .,2008) .

2.3.1 History of apoptosis German scientist Carl Vogt was first to describe the principle of apoptosis in 1842. In 1885, anatomist Walther Flemming offered a more precise description of the process of programmed cell death ( Karam , 2009). However, it was not until 1965 that the topic was resurrected. Kerr had

initially

used

the

term

programmed

cell

necrosis

( Kerr et al.,1972) ,but in the article, the process of natural cell death was called apoptosis.

Kerr

received

the Paul

Ehrlich

and

Ludwig

Darmstaedter prize on March 14, 2000, for his description of apoptosis (O'Rourke and Ellem, 2000). The word ´apoptosis´ is of Greek origin, meaning "falling off or dropping off", in analogy to leaves falling off trees 26

or petals dropping off flowers. Although most of the observable events that define apoptosis and its role in embryonic development were well documented as early as the 1950s, the term apoptosis was not known until another 20 years (Kerr et al., 1972). Apoptosis is a multi-step, multi-pathway cell-death programmed that is inherent in every cell of the body. In cancer, the apoptosis cell-division ratio is altered. Cancer treatment by chemotherapy and irradiation kills target cells primarily by inducing apoptosis.

2.3.2 bCL2 and apoptosis 2.3.2.1 Apoptosis regulator bcl-2 : bcl-2 (B cell lymphoma/leukemia-2) gene, an oncogenic that located on chromosome 18 (18q 21.3) .This gene

Chapter Two


in follicular lymphoma is frequently linked to an immunoglobulin locus by the chromosome translocation (14;18), was the first example of an oncogenic that inhibits cell death rather than promoting proliferation (Chao and Korsmeyer , 1998). When homologues of bcl-2 had been identified, it became apparent that a bcl-2 family of proteins could be defined by the presence of conserved sequence motifs known as bcl-2 homology domains (BH1 toBH4). In mammals, up to 30 relatives have been described of which some belong to a group of pro-survival (anti-apoptotic) members and others to a group of pro-apoptotic members(Figure 2-1). Most of the bcl-2 family members have a hydrophobic trans- membrane domain (TM) in its terminus that enables the insertion of the protein into membranes and variable amount of BH regions (Faderl et al., 2002). A. Pro-survival (anti-apoptotic) members:

27

In addition to bcl-2 itself, there are a number of other pro- survival proteins, e.g. bcl-XL; L stands for ´long´.

It renders cells resistant to

apoptotic cell death upon deprivation of growth factors, bcl-w, A1, and Mcl1, which all possess the domains BH1, BH2, BH3, and BH4 (Cory and Adams, 2002). B. Pro-apoptotic members: The pro-apoptotic group of bcl-2 members can be devided into subgroups: 1. The Bax-subfamily consists of Bax, Bak and Bok that all possess the domains BH1, BH2, and BH3 and TM. 2. Bad consists of BH1, BH2, and BH3 but no TM.


Chapter Two

3. The bcl-XS ; S stands for ´short´, it counters the resistance to apoptotic cell death provided by bcl-2(Vogelstein et al., 2000; Vousden and Lu, 2002; Tian et al., 2007). 4. BH3-only family. BH3-only family of proteins includes those of the bcl-2 family proteins, which contain only a single BH-domain. The BH3-only family members play a key role in promoting apoptosis (Cory and Adams, 2002).The BH3only family members are Bad, Bim and others. Various apoptotic stimuli induce expression and/or activation of specific BH3-only family members, which translocate to the mitochondria and initiate bax/bak-dependent apoptosis (Michael et al., 2008) .

28

Figure (2-1) bCL2 family ( Chao and Korsmeyer ,1998)

2.3.2.2 Function bcl2 -Homology ( BH) is a family of evolutionarily related proteins, these proteins govern mitochondrial outer membrane permeabilization (MOMP).

Active cell suicide (apoptosis) is induced by events such

as growth factor with drawl and toxins. It is controlled by regulators, which Chapter Two


have either an inhibitory effect on programmed cell death (anti-apoptotic) or block the protective effect of inhibitors (pro-apoptotic) ( Vaux,1993 ; Milliman et al.,1996) . Many viruses have found a way of countering defensive apoptosis by encoding their own anti-apoptosis genes preventing their target cells from dying too soon. The bcl-2 family has a general structure that consists of a hydrophobic helix surrounded by amphipathic helices. Many members of the family have trans- membrane domains. The site of action for the bcl-2 family is mostly on the outer mitochondrial membrane. Within the mitochondria are apoptogenic factors (cytochrome c, Smac/Diablo homolog, Omi) that, if released, activate the executioners of apoptosis, the caspases (Fesik and Shi, 2001) .Depending on their function, once activated, bcl-2 29

proteins either promote the release of these factors, or keep them sequestered in the mitochondria. Whereas the activated pro-apoptotic bak and/or bax would form mitochondrial apoptosis induced channel( MAC) and mediate the release of cytochrome c, the anti-apoptotic bcl-2 would block it, possibly through inhibition of bax and/or bak ( Dejean et al., 2006).

2.3.3 Apoptotic Mechanism The pathways of apoptosis are extremely complicated, where energy dependent flow of molecular events takes place (Figure 2-2). Mainly two types of apoptotic pathways such as intrinsic and extrinsic(or death receptor) have been well described that involve a number of proteins. Another pathway of apoptosis has also been recognized which involves perforin/ granzyme-A or B (released by cytotoxic T lymphocytes and NK cells: perforin/granzyme induced apoptosis). It has been found that granzyme Ainduced apoptosis is a caspase-independent pathway through single stranded DNA damage (Martinvalet et al., 2005). Review of Literature

Chapter Two

Both intrinsic and extrinsic pathways of apoptosis are associated and influence each other (Igney and Krammer, 2002).All the three (intrinsic, extrinsic and granzyme B -induced) pathways of apoptosis come together on the same point putting to cell death by the activation of caspase-3/7 followed by

cell

shrinkage, chromatin

condensation

and

fragmentation

of

chromosomal DNA, degradation of nuclear as well as cytoskeleton proteins etc. (Elmore, 2007). Generally, caspases occur in the cells in an inactive form (i.e.,pro caspase) ,other caspases leading to amplification of apoptotic signaling pathway followed by cell death due to their proteolytic activity. ….Caspases are widely expressed in an inactive pro-enzyme form in most cells and once activated can often activate other pro-caspases allowing

30

initiation of a protease cascade. Some pro-caspases can also aggregate and auto activate.

Figure (2-2) :Mechanism of apoptosis( Rajesh et al.,2009)

Chapter Two


This proteolytic cascade, in which one caspase can activate other caspases, amplifies the apoptotic signaling pathway and thus leads to rapid cell death.

2.3.3.1 Extrinsic pathway of apoptosis Extracellular signals may include toxins (Popov et al., 2002), hormones, growth factors and nitric oxid (Brune,2003).Exposure of normal human keratinocytes to UV radiation induces the extrinsic apoptotic pathway, which is initiated by the cell surface death receptors, such as TNF1 receptor (p55 or CD120a), Fas (CD95/APO-1), DR3(Apo3), DR4 and DR5 (Haupt et al., 2003; Yan and Shi, 2005; Elmore, 2007). UV-B-induced apoptosis may be triggered by ligand dependent (Caricchio et al., 1998) or 31

ligand-independent (Rehemtulla et al., 1997) Fas clustering, with consequent caspase activation (Clement et al., 2007). Activation of death receptor is followed by the formation of death inducing signaling pathway complex (DISC), which is composed of adaptor molecule FADD (Fas associated death domain) and caspase-8 (Boldin et al., 1996). FADD is linked with their N- terminal death caspase-8 zymogens by means of hemophilic interface with effector domains (DEDs) (Boatright and Salvesen, 2003). With the formation of DISC, caspase-8 is activated, which either directly cleaves and activates the effector caspases, or indirectly activates the downstream caspases through the cleavage of BH3-only protein Bid, leading to involvement of intrinsic pathway of apoptosis (Luo et al., 1998).

2.3.3.2 Intrinsic pathway of apoptosis Following DNA damage, an increased level of Bax and/or a decreased level of bcl-2 may permeabilize the mitochondria to release pro-apoptotic factors, such as cytochrome- c, which trigger subsequent activation of Chapter Two


Pro-caspase-9, followed by downstream apoptotic effectors (Crompton, 2000). It has also been found that in response to DNA damage, activation of caspase-2 is required before mitochondrial permeabilization and release of cytochrome-c (Guo et al., 2002; Lassus et al., 2002; Vakifahmetoglu et al., 2006). Activation of caspase-2 might occur without processing of the precursor molecule. However, oligomerization is an important step for caspase-2 activation (Norbury and Zhivotovsky, 2004). Expression of cyclin-D3 and caspase-2 in human cells potentially induces apoptosis, compared with expression of caspase-2 alone, which shows the involvement of cyclin-D3 in caspase-2 activation (Mendelsohn et al., 2002). After releasing cytochrome32

c, it binds to apoptotic protease activating factor Apaf-1 and forms a 7-span symmetrical active complex ‘apoptosome’ in nucleotide dATP/ATP dependent manner (Zou et al., 1999). The apoptosome subsequently recruits procaspase-9 into its central region to form an active holoenzyme, which further activates downstream executioner caspases, such as caspase-3/7 that leads to PCD (Jiang and Wang, 2004). It has been found that in mammalian cells, caspase activity is also stimulated by one of the pro-apoptotic mitochondrial proteins Smac/Diablo, and Omi/HtrA2, which promote apoptosis by interfering the action of inhibitor of apoptosis (IAP) family protein (e. g., survivin) (Verhagen et al., 2002; Song et al., 2003). The inhibition of apoptosis through IAPs family is not universal, as survivin is unable to prevent the induction of apoptosis in ovarian cancer cells by Smac (McNeish et al., 2005). In addition to caspase activator proteins, some other molecules unrelated to caspase activation such as AIF (apoptosis inducing factor) (Susin et al., 1999) and Endo G Chapter Two


(endonuclease G) (Li et al., 2001) has also been found to be released from mitochondria that cause apoptosis by DNA fragmentation and subsequent chromosomal condensation. An evidence suggests that Bax/Bak can also be localized in the endoplasmic reticulum (ER) and gets activated in response to ER stress, leading to calcium depletion and murine caspase-12 activation (Scorrano et al., 2003). The result of these biochemical changes is the appearance of morphological changes in the cell. So apoptosis is a morphological phenomenon, while can be viewed with the assistance of light or, preferably, the electron microscope (Figure :2-3).

33

Figure (2-3):Morphological changes of apoptotic cells(Chiarugi and Moskowitz ,2002) Chapter Two


These morphological changes include: 1. Cell shrinkage and rounding are shown because of the breakdown of the proteinaceous cytoskeleton by caspases. 2. The cytoplasm appears dense, and the organelles appear tightly packed. 3.Chromatin undergoes condensation into compact patches against the nuclear envelope (also known as the per- inuclear envelope) in a process known as pyknosis, a hallmark of apoptosis. (Susin et al .,2000; Madeleine et al .,2001). 34

4. The nuclear envelope becomes discontinuous and the DNA inside it is fragmented in a process known as karyorrhexis. The nucleus breaks into several

discrete chromatin

bodies or nucleosomal

units due

to

the

degradation of DNA (Nagata, 2000). 5. The cell membrane shows irregular buds known as blebs. 6. The cell breaks apart into several vesicles called apoptotic bodies, which are then phagocytosed. Apoptosis progresses quickly and its products are quickly removed, making it difficult to detect or visualize. During karyorrhexis endonuclease

activation leaves short DNA

fragments, regularly spaced in size. These give a characteristic "laddered" appearance..on agar gel..after electrophoresis.. Tests..for DNA..laddering differentiate apoptosis from ischemic or toxic cell death (Iwata et al .,1996). Chapter Two

2.4


Flow cytometry

Flow cytometry is now a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in heterogeneous cell populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. It allows simultaneous multi-parameter analysis of single cells. It is predominantly used to measure fluorescence intensity produced by fluoresent-labelled antibodies detecting proteins or ligands that bind to specific cell-associated molecules, such as DNA binding by Propridium Iodide. Cells are then analysed on the flow cytometer. In the flow cytometer, 35

the sample is hydrodynamically focused to a tiny stream of single cells. Laser light is directed onto the sample as it flows through the chamber. …There are a number of detectors which detect the light scattered from the cells/particles as they go through the beam. There is one in front of the light beam forward scatter ( FSC) and several side on to it side scatter (SSC).Fluorescent detectors are used for the detection of fluorochromes themselves. Particles/cells passing through the beam will scatter the light, which is detected as forward scatter and side scatter. The combination of scattered and fluorescent light is detected and analyzed. forward scatter correlates with the cell size and side scatter depends on the density of the particle/cell (i.e. number of cytoplasmic granules, membrane size) . In this manner cell populations can often be distinguished based on their difference in size and density. Fluorochromes used for detection/staining will emit light when excited by alaser with the corresponding excitation wavelength. These particles/cells can be detected individually and the data analyzed .Flow cytometric analysis FCM techniques have been used to study apoptotic events and their temporal Chapter Two


course in different cell types in response to various triggers ( Schmid et al.,2007) .The studied parameters include changes in the distribution of phospholipids across the cell membrane (Troiano et al.,2007). Several reports have shown increased exposure of PS on the outer leaflet of the plasma membrane of different cell types undergoing apoptosis, including lymphocytes, thymocytes, and tumor cell lines of lymphoid and of neural origin (Fadok etal.,1992;Homburg etal.,1995;Rimon et al.,1997). …Differentiating between CLL cells of either apoptosis or necrosis was carried out using Annexin V-FITC kit. Annexin-V, Ca2+ -binding protein was first described by (Reutelingsperger et al.,1985) as a vasculature36

derived protein with strong anticoagulant properties. Annexin-V binds with high affinity to PS; commercially available annexin-V conjugated to fluorochromes is used in an apoptotic detection assay by FCM analysis .

2.5 Chemotherapy Chemotherapy is a leukemia treatment that uses drugs to stop cell division (National Cancer Institute,2007). There are different types of chemotherapy, each with its own target and goal. Some aim to wipe out the cancer while others try to stop cancer from progressing while treating any symptoms (Rai et al.,2005). The main focus of CLL chemotherapy has tended to be symptomatic relief with the lowest risk of health complications from treatment since there has not yet been a associated with longer survival than partial or no remission in response to therapy. The goal of chemotherapy in regards to CLL is to obtain high rates of complete response( CR )in patients and extend remission as long as possible in the hope that overall survival (OS) rates will also increase (Wierda , 2006). Chapter Two


2.5.1 Alkylating agents Chlorambucil Alkylating agents result in some symptomatic improvement, but not all symptoms disappear completely and survival is not prolonged. Comparative studies affirm this information with partial response rates as high as 72% but chances of complete remission as low as 4% ( (Palma et al., 2006). Alkylating agents are a group of drugs that hinder the process of cell replication (Abbott, 2005).They modify DNA by inserting an alkyl hydrocarbon group. When this group is present, the double helix DNA cannot unwind.

37

As a result, the targeted cells cannot reproduce and eventually die (Byrd, 2006). Chlorambucil, which is the most common alkylating drug for CLL, has been shown to have an over 50% partial response rate but no significant increase in complete cure rates (Canadian Cancer Society, 2006). Unfortunately, the side effects of alkylating therapy overshadow the potential for symptomatic relief . Most alkylating agents are derivatives of poisonous substances such as mustard gas and are understandably associated with detrimental effects. The first issue with the drugs is the non discriminatory nature of their treatment. Alkylating agents target all fast growing cells not just cancerous B lymphocytes. This leads to lowered blood cell numbers overall (red, white, and platelets) which increases risks of infection, bleeding, bruising, anemia and fatigue in patients. Alkylating drugs also attack intestinal cells causing queasiness, appetite loss and vomiting. Other side effects include hair loss and mouth sores. Alkylating chemotherapy also increases risks of secondary cancers developing (Abbott, 2005). Alkylating chemotherapy is associated with extensive side effects including increased risks of infection and development of additional malignancies which must be weighed against Chapter Two


the potential for partial symptomatic improvement. With alkylating treatment of CLL “the quality of response is low ( 0%-31%) and the duration of responses usually short (2-18 months)´´ (Yee and O’Brien, 2006). While partial relief of some CLL symptoms may be obtained with alkylating therapy, it comes at a high risk of serious health complications and lowered quality of life.

2.5.2 Purine nucleoside analogs Fludarabine, Cladribine Purine analogs are a newer form of chemotherapy drugs that work in a slightly different way with different 38

results from alkylator therapy (Rai et al.,2000).Purine analogs act as inhibitors to DNA replication. They either replace or inhibit CLL enzymes such as DNA polymerase which is essential to DNA replication (Palma et al., 2006). Without such enzymes DNA reproduction is halted and apoptosis is stimulated, killing off cells. The main purine analogs used in CLL treatment are Fludarabine and Cladribine. Studies show that compared to alkylator therapies, Fludarabine and Cladribine have higher complete response( CR) rates, longer periods of CLL dormancy and greater symptomatic response. However, purine analogs show no significant improvement in overall survival rates (Dighiero et al., 1998; Eichhorst et al., 2009). Studies tested alkylator chlorambucil and purine nucleoside Fludarabine as monotherapies and also in combination (Rai et al.,2000; Wierda, 2006) . …The combination therapy proved too toxic and could not be continued, but Fludarabine yielded CR rates more than 15% higher than Chlorambucil with remissions usually lasting 25 months. However, overall survival rates were not increased in comparison to alkylator chemotherapy trials.

Chapter Two


Fludarabine may also cause CLL to develop into a less treatable form of the disease CLL cells without a certain protein (p53 which is an important gene responsible for apoptosis) gene are immune to Fludarabine. Therefore, when Fludarabine wipes out CLL cells, resistant CLL cells are left behind that can only be treated with harsher therapies that have increased risks of infection and other severe side effects (Hamblin, 2001). Purine analogue chemotherapy has a long list of side effects including bone marrow suppression, severely low lymphocyte, platelet and neutrophil levels, immunosuppresion associated with secondary malignancies and high rates of infection by a variety of opportunistic pathogens (Palma et al., 39

2006). With increased risk of immuno- and myelosuppression, infection, anemia, secondary cancers and CLL mutations, alkylating agents provide temporary

symptomatic relief but also increase chances of detrimental

health problems and ultimately, shortened life (Byrd, 2006). Although Purine analog therapy may yield higher response rates than alkylator therapy , it also shows increased severity and occurrence of negative side effects. It is clear that chemotherapy adds more health problems to the already heavy load CLL patients bear. Chemotherapy can provide temporary symptomatic relief but no increase in overall survival. It increases chances of developing serious health complications that can decrease survival length of patients. As a result it can be concluded that treatment based on chemotherapy is not the source of a cure for CLL (Tam, 2008).

Chapter three

MATERIAL AND METHOD

3.1.Material 3.1.1. Laboratory Equipment The general laboratory equipment that were employed in the study are listed in table (3.1).

.

Table ( 3.1)General laboratory equipments Equipment

Company / Country

Autoclave Digital camera

SES Little Sister / England Mercury / China 0B

Electrophoresis power supply

pelex/ France

40

Eppendorf centrifuge

MIKRO 120 / Germany

Flow cytomertery

Apogee flow system /Uk

Horizontal Electrophoresis unit

pelex/ France

Magnetic stirrer with hot plate

Lassco /India

Oven

Osaw / India

pH meter

Radiometer / Denmark

Sensitive balance

Sartorius /Germany

Shaker

Finpcr /Korea

Universal centrifuge

Humax 4k / Germany

Veriti 96 well thermal cycler

Applied biosystem/USA

Vortex

Griffin / England

Water bath

Techne DRI-Block /UK

MATERIAL AND METHOD

Chapter three 3.1.2Chemical Materials

The chemical materials that were used in the study are listed in table (3.2). Table ( 3.2). Chemical materials. Chemical Material

Company / Country

Agarose

promega / USA

Ammonium chloride(NH4Cl)

Fluka/Germany

Annexine V-FITC

Exbio/Korea

41

Chemical Material

Company / Country

Diatrizoate

Sigma/USA

DNA ladder marker (100 bp)

Promega/USA

DNA loading dye

promega / USA

DNTPS (Mix)

Promega/USA

EDTA

BDH/England

Ethidium bromide (EtBr)

BDH/England

Ficoll solution

Sigma/USA

Mertizoate

Sigma/USA

Phosphate buffer saline (PBS)

Schucrarlt/Germany

PI

Exbio/Korea

Potassium bicarbonate(KHCo3) Fluka/Germany Primers

Alpha DNA/ Canada

Profi Taq PCR premix

Bioneer /South Korea

Tag polymerase

Promega/USA

TBE buffer(10 x)

Sigma/USA

Chapter three

MATERIAL AND METHOD

3.2 Methods 3.2.1 Specimen collection 3.2.1.1 Patient selection Patients

were

selected

according

to

clinical

and

laboratory

examination. Aquestionnaire form was filled for each patient; it included

42

name, age, job, family history, geographic origin, date of diagnosis, type of leukemia and last chemotherapy administration. 3.2.1.2 Blood sampling Three

ml of blood was collected by vein

puncture from 30

cases(CLL) after treatment who were admitted to the center of hematology /Yarmouk hospital from September 2012 till January 2013 .They were clinically diagnosed by the consultant medical staff of the center. Each collected blood sample was dispensed into an EDTA tube, 15 apparently healthy (blood donors)were also included as a control group.

3.2.2 Isolation of lymphocytes 3.2.2.1 Preparation of solutions A . Phosphate buffer salin (PBS) The buffer was supplied as a sterile powder ready to dissolve in one liter. Ten gm of PBS powder was dissolved in 1 liter of distilled water. The pH was adjusted to 7.4 , the solution was autoclaved (121°C ,15 pound/inch² for 20 min)and stored at 4 C° until being used. B .Lyses buffer The buffer was prepared according to (Kaplan ,1982). It contained the following per liter of distilled water: -Ammonium chloride(NH4Cl) 8.29gm.

MATERIAL AND METHOD

Chapter three -Potassium bicarbonate (KHco3)1gm. -EDTA 0.037gm.

The solution was autoclaved (121 C°,15 pound /inch² for 20 min.

3.2.2.2

Isolation Protocol of Lymphocytes (Hokland and Heron 1980). 43

1. Two milliliters of blood sample were added to an anticoagulant tube with potassium EDTA and was diluted 1:1 ratio with phosphate buffer saline (PBS, pH 7.2) to washout and resuspend the separated lymphocyte for different analytical procedures. 2. Four milliliters of the diluted blood sample were carefully layered (not mixed) on the top of four milliliters of lymphocyte separation media (Ficoll), which was dispersed in a 15 milliliter siliconized glass conical centrifuge tube. (Lymphocyte separation medium is an aqueous solution containing Ficoll and sodium metrizoate or diatrizoate at apre determined density of 1.077 g/ml at 25 ºC.). 3. The tube was centrifuged at (2700 rpm) at room temperature for 30 minutes . After centrifugation, the lymphocytes formed a creamy-web like-layer at the interface of the blood plasma and the separation medium(Figure 3- 1). 4. The separated layer lymphocytes were aspirated (not disturbed) by Pasteur pipette and transferred into another siliconized centrifuge tube. 5. The aspirated lymphocytes were washed by lyses buffer first time if there were more RBCs in the tube for lysing it, and centrifuged for 15 minutes at (2700 rpm) until a pellet was formed. The supernatant was discarded. 6. The pellet was washed by PBS (2ml), centrifuged for 15 minutes at (2700 rpm) until a pellet was formed. The supernatant was discarded. Chapter three

MATERIAL AND METHOD

This procedure was repeated for twice to washout the lymphocyte suspension. 7. Finally, the lymphocyte pellet was ready to be used in the planned experiment.

44

Lymphocyte layer

Figure( 3-1):The isolation of lymphocyte layer

3.2.3 Detection of apoptosis in the isolated lymphocytes 3.2.3.1. Principle This study was designed to qualify apoptosis induction after treatment with chemotherapy by using Human Annexin V-FITC apoptosis detection kit( ExBio Praha). Apoptosis is a regulated cell death process characterized by morphological and biochemical features occurring at different stages. One of the plasma membrane alterations during apoptosis was the translocation of MATERIAL AND METHOD

Chapter three

phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Detection of such membrane changes by Annexin V has been suggested as a suitable assay of early apoptotic cells. Annexin V is a protein which belongs to a member of a Ca2+ -depended phospholipids binding family of P

P

45

proteins. Fluorescently labeled Annexin V can be applied for direct quantification of apoptotic cell by using appropriate protocols. Since PS membrane translocation also occurs during necrosis, Annexin V was not an absolute marker of apoptosis; it was often used simultaneously with appropriate dyes such as propidium iodide (PI) to counter stain dead cells. AnnexinV-FITC employed in cytometry was performed according to the(human Annexin V-FITC Kit-ExBio Praha). This kit includes binding buffer, Annexin V-FITC, and Propidium iodide (Dillon et al., 2001; Jitkaew et al., 2009). 3.2.3.2 Procedure (List in appendex) 1. The lymphocyte pellet isolated in section (3.2.2.2) was resuspended with diluted binding buffer (10x)in deionized water prior use(1x), preparing a sufficient volume of cell suspension (180 microlitters). 2. Ten microlitters of Annexin V-FITC and ten micro litters of Propidium iodide (0.1mg/ml) were added to each 180 microlitters of cell suspention and gently mixed. 3. The stained cells were incubated for 15 mins in dark at room temperature. 4. The cells were then centrifuged at 2000 rpm for 5 mins, the supernatant was discarded and the pellet resuspended with 180 µl of binding buffer(1x). 5. The stained cells were analyzed by flowcytometry .

MATERIAL AND METHOD

Chapter three

3.2.4 DNA Extraction Extraction was accomplished according to Qiagene DNA purification kit protocol.

3.2.4.1 Kit components • Protease enzyme or proteinase K. 46

• Lysis buffer(AL). • Ethanol (96-100%) • Washing buffer(1)(AW1) • Washing buffer(2)(AW2). • Elution buffer(AE). 3.2.4.2 Procedure of DNA Extraction: 1. Twenty μl Protease (or proteinase K) enzyme was pipette into the bottom of a 1.5 ml micro centrifuge tube. 2. 200 μl sample(blood) was added to the micro centrifuge tube. 3. 200 μl Buffer AL was added to the sample and was mixed by pulsevortex for 15 sec. 4. The mixture was incubated at 56°C for 10 min. 5. The tube was centrifuged to remove drops from the inside of the lid. 6. 200 μl ethanol (96–100%)was added to the sample tube , mixed again by pulse-vortex for 15 sec , and centrifuged at 2000 rpm for 1 min to remove drops from the inside of the lid. 7. The mixture from step 6 was carefully applied to the mini spin column (in a 2 ml collection tube) without wetting the rim, the cap was closed and centrifuged at 6000 xg(8000 rpm) for 1 min. The mini spin column was placed in a clean 2 ml collection tube ; the tube containing the filtrate was discarded. Chapter three

MATERIAL AND METHOD

8. The mini spin column was opened and 500 μl buffer AW1 was added without wetting the rim; the cap was closed and centrifuged

at 6000 x g

(8000 rpm) for 1 min. The mini spin column was placed in a clean 2 ml collection tube ,and the collection tube containing the filtrate was discarded.

47

9. The mini spin column was opened and 500 μl buffer AW2 was added without wetting the rim; the cap was closed and centrifuged at full speed (20,000 x g; 14,000 rpm) for 3 min. 10. The mini spin column was placed in a clean 1.5 ml micro centrifuge tube, and the collection tube containing the filtrate was discarded. Carefully the mini spin column was opened and 200 μl buffer AE or distilled water was added. 11. The mixture was incubated at room temperature (15–25°C) for 1 min, and then centrifuged at 6000 x g (8000 rpm) for 1 min.

3.2.5 : DNA Checking: (Sambrook et al., 1989) 1.

DNA integrity: DNA samples (7 µl DNA+1-2µl loading dye) were electrophoresed

through 1% agarose gel for 1 hour/ 70 volt. The gel was then stained with 12µl of ethidium bromide (5mg/ml concentration)and visualized under a UV transilluminator. The integrity of the DNA was jugged when a single band appeared but no smear band (degrade DNA).

2.

DNA concentration: DNA dilution was made and applied (1:10) for spectrophotometry

at 260 A°. The DNA concentration was calculated according to Sambrook et al., (1989). O.D 260 nm × Dilution factor × 50 µg/ml = µg/ml Chapter three

3.

MATERIAL AND METHOD

DNA purity:

1:10 DNA dilution was applied for spectrophotometry at 260 A° once and then at 280 A°. The purity was calculated as follows: Pure DNA = O.D A 260 / A 280 = ~ 2 (1.8 – 2). 48

3.2.6 Agarose Gel Electrophoresis Reagents Agarose 1 X TBE buffer (Trice borate EDTA). Loading dye (bromophenol blue in 1% glycerol). DNA marker(100 base pair). Ethidium bromide (5mg / ml).

3.2.6.1 Protocol 1-Preparation of 1x TBE buffer (Sambrook et al., 1989) The solution was prepared by adding 900 ml of distilled water to 100 ml of TBE (10 x),forming 1 liter of( 1x) TBE buffer . 2-Preparation of agarose gel.  Additon of 50 ml of 1 X TBE ( pH 8.0) in a beaker.  Addition of 0.5 gm (or 1 gm for 2%) agarose to the buffer.  The solution was heated till boiling (using water bath)  The solution was allowed to cool down at 50-60°C.  Two concentration of a garose gel was prepared

(1% and 2% ) as

required.

Chapter three

MATERIAL AND METHOD

• Casting of the horizontal agarose gel:  The gel was assembled to a casting tray and the comb was positioned at one end of the tray.

49

 The agarose solution was poured into the gel tray after both edges were sealed with tapes and the agarose was allowed to gel at room temperature for 30 minutes.  The comb was carefully removed and the gel was replaced in an electrophoresis chamber.  The chamber was filled with TBE-electrophoresis buffer until the buffer reached 3-5 mm over the surface of the gel. • Loading and running DNA in agarose gel  DNA was mixed with loading dye (ratio 3:1) and loaded in the wells of the agarose gel.  The cathode was connected to the well side of the unit and the anode to the other side.  The gel was run at 70 volt. until the loading dye migrated to a suitable distance from the well. The DNA was observed by staining the gel with ethidium bromide and viewed under a UV transilluminator (302 nm).

Chapter three

MATERIAL AND METHOD

3.2.7 Polymerase Chain Reaction ( PCR): The PCR reaction was performed using the specific bcl2 gene primers as in figure (3-2) according to ( Loro et al. ,2005) which were aligned with 50

whole bcl2 genome using blast software. Lyophilized primers (Alpha DNA/Canada) were dissolved in

sterile distilled water to give a final

concentration of (10 p mol/µl).The sequences of these primers were listed in table (3.3). Sequence No. of primer

Forward

of primers Reverse

molecular size of product( bp)

P1

5´ cctcgtccaagaatgcaa3´

5´ gctgggaggagaagat 3´

291

P2

5´ cccggcgacgacttct 3´

5´ agagcccacccgcactc 3´

367

Table (3. 3) Primer sequence for amplification bcl2 gene ( Loro et al. ,2005)

p1 ( 291) p2(367)

Figure(3-2) Schematic diagram of the bcl2 gene &the PCR primers used Chapter three

MATERIAL AND METHOD

3.2.7.1 PCR working solutions

51

Taq ready mix supplied in a 2X concentrate reaction buffer, ( Go Taq® Green Master Mix(Ready-to -use) (Promega /USA) included Taq DNA polymerase ,dNTPs, MgCl2 and reaction buffer (PH 8.5 )) offered the standard concentrations of Taq DNA polymerase of 1.5 U and 200 µM of dNTPs in each reaction. Ten pmol of each primer were used .The primers concentration represent the optimal concentration. A decrease in the primer concentration leads to weak PCR product while an increase could result in the formation of primer dimer artifact leading to misinterpretation of results (Saiki, 1988). The PCR based techniques do not require highly purified DNA preparations as it works well with partially purified DNA samples (Edward

et al., 1991; McPherson and Moller, 2001). However DNA

extraction may contain inhibitory compounds, like detergents used in cell lysis and protein denaturation in addition to other inhibitory compounds that could interfere with PCR leading to reaction failure (McPherson and Moller, 2001). PCR reaction materials are listed in tables (3.4) for P1 and (3.5) for P2. Table (3.4) Master mix components of bcl2 PCR (P1)

Chemicals H2O Primer 1/Forward Primer 1/Reverse Green master mix

bcl2 gene/P1 Volume (µl) 8.5 µl 0.5µl 0.5µl 12.5 µl

DNA Final Volume

3 µl 25 µl

Concentration 10 pico moles 10 pico moles 1.5 U &200µM of dNTPs

1-3ng/µl

Chapter three

MATERIAL AND METHOD

bcl2 gene /P2 52

Chemicals H2O Primer2/Forward

Volume(µl) 8.5 µl 0.5 µl

Concentration

Primer 2/Reverse

10 pico moles

Green master mix

0.5µl 12.5 µl

DNA Final Volume

3 µl 25 µl

1-3ng/µl

10 pico moles 1.5 U &200µM of dNTPs

Table (3.5) Master mix components of bcl2 PCR (P2)

3.2.7.2 PCR protocol A successful PCR program depends on the reaction conditions including reagents, temperature and the prevention of contamination. previous study indicates that PCR is sensitive to reaction condition and that the optimization of these conditions is necessary to reach the highest specificity and product yield (Williams et al., 1990). Standard amplification conditions were applied in PCR with primer sequences. The annealing temperature in which primers hybridize to complementary sequences on the template DNA is perhaps the most critical in PCR programming. The annealing temperature for PCR primers is based on melting temperature (Tm) calculations. Tm is the temperature at which half of DNA strand is denatured. A Tm calculation for PCR primers is based on guanine and cytosine (G+C) content. The annealing temperature is usually below the Tm in 2-12 °C (Newton and Graham, 1997).

Chapter three

MATERIAL AND METHOD

53

Optimization of amplification was performed under the following conditions listed in tables (3.6) for P1 and (3.7) for P2.

No. 1.

PCR with bcl2 gene/P 1 Steps Temperature Time Initial denaturation 95 °C 3 min

2. 3. 4. 5.

Denaturation 2 Annealing Extension 1 Final extension

95 °C 59 °C 68 °C 68 °C

22sec 30sec 25sec 5 min

No. ofcycles 1 cycle 35 cycle

1 cycle

Table (3.6) The PCR program for P1 region of bcl2 gene amplification

No. 1. 2. 3. 4. 5.

PCR with bcl2 gene/P2 Steps Temperature Time Initial denaturation 95 °C 3 min Denaturation 2 95 °C 22sec Annealing 63°C 30 sec Extension 1 68 ºC 25 sec Final extension

68 ºC

5 min

No. of cycles 1 cycle 35 cycle

1 cycle

Table (3.7) The PCR program for P2 region of bcl2 gene amplification We used different temperature to get an optimal temperature for annealing, the best temperature being 59°C for ( P1) and 63 Cº for ( P2).

Chapter three

MATERIAL AND METHOD 54

Negative control was also used in each PCR reaction to confirm the accuracy of the PCR reaction. PCR products were electrophoresed through 1.5 % agarose gel, 70 volt for 1 hour. The gel was stained with ethidium bromide (5mg/ml) and visualized under UV trans-illuminator to detect the presence of P1 and P2 bands. The presence of 291 bp refers to P1 band while the 367 bp belongs to P2 band. A DNA ladder (100 bp) was used to estimate the molecular size of the bands.

3.2.7.3 PCR products sequencing The PCR products (10 samples) of the analyzed bcl2 gene regions and primers were sent to the Macrogen Company(USA) for sequencing . …The PCR reaction volum was 50µl, using profi Taq PCR premix (Bioneer/Korea). The components were listed in table (3.8).

Chemicals H2O Primer 1/Forward Primer 1/Reverse DNA Final Volume

bcl2 gene/P1 &P2 Volume Concentration (µl) 42µl 1µl 10 pico moles 1µl 10 pico moles 6 µl 1-3ng/µl 50 µl

Table (3.8) ProFi Taq PCR premix components of bcl2 gene( P1&P2)

Chapter three

MATERIAL AND METHOD

55

The ProFi Taq PCR premix condition were listed in table (3.9).

No. 1. 2. 3. 4. 5.

ProFi Taq PCR premix with bcl2 gene/P1&P2 Steps Temperature Time No. of cycles Initial denaturation 95 °C 5 min 1 cycle Denaturation 2 95 °C 15-20sec 35 cycle Annealing 63°C 30 sec Extension 1 68 ºC 25 sec Final extension

68 ºC

5 min

1 cycle

Table (3.9) The ProFi Taq PCR premix condition for P1& P2 regions of bcl2 gene amplification The sequences of these samples were compared with the information in gene bank of the national center for biotechnology (NCBI) of standard bcl2 gene, using (Mega -5) software for detection of the mutations in bcl2 gene.

3.8 Statistical Analysis The Statistical Analysis System- SAS (2010) was used to study the effect of infection on the study´s parameters. The t-test was used to compare these results.

Chapter Four

Results and Discussion

4. 1: Subject data 56

A total of 30 EDTA blood samples were collected from chronic lymphocytic leukemia (CLL) patients after treatment and fifteen EDTA blood samples were collected from apparently healthy young people(control) group ,as well.

4.1.1 Age The age of patients were ranged from (35 years to 75 years). Reports show that the prevalence of CLL mostly occurs in older people, but can theoretically occur in any age group, most often affects adults over the age of 55. It sometimes occurs in younger adults, but it almost never affecting children. Two-thirds of the affected people are men (Jameson e t al., 2005). …The incidence of most tumors rises strikingly with age (Newell et al., 1985). Many of

the assays detect an increase in mutation frequency with

age in blood cells (Strauss and Albertini , 1979; Turner et al., 1985).

4.2 Flow cytometry Annexin V-FITC Apoptosis Detection To detect intracellular defect occurs by bcl-2 gene mutation in individual cells, cells were incubated with Annexine V-FITC kit .This is a reliable method of cellular detection in flow cytometry which makes it possible to obtain a higher number of labelled cells and agreat mean fluorescence intensity (MFI). Different flow cytometric (FCM) techniques have been developed for the characterization and quantification of the various cellular events during apoptosis using Annexin V-FITC kit. bcl-2 family protein expression was measured in patients after treatment and in healthy groups. Tables(4-1;4-2) shows how apoptotic and viable cells were discriminated using changes in FSC and SSC.These gates were subsequently Chapter Four


57

used to determine bcl-2 protein expression in the two separate populations of cells (Figures 4-1 , 4-2).The different labeling patterns in this assay identify the different cell populations: Region-A:necrotic cells (PI-positive/annexin V-negative) . Region-B: late apoptosis (secondary necrosis) (PI-positive/annexin Vpositive). Region- C: vital cells (PI-negative/ annexin V-negative ) . Region-D:apoptotic..cells(PI-negative/annexinV-positive). …The investigated apoptotic cells were divided into two groups

A: for

patients after treatment ; B : for control samples (Healthy).

A

FL2

B

FL1

C D

Figure (4-1) Annexin V expression for apoptosis analysis in

control

samples . FL1(Horizantal) and FL2 (Vertical).,showed intensity of Annexien V-FITC and PI staining, respectively The different labeling patterns in this assay identify different cell populations, e.g. ; region C: vital cells (PInegative/ annexin V-negative) Chapter Four


58

FL2

A

FL2

A

B

B

FL1

c

D

FL1 C

D

Figure (4-2) Annexin V expression in CLL patients and healthy samples induced apoptosis analysis ( apoptosis cells-stain cells).FL1(Horizantal) and FL2 (Vertical) showed intensity of Annexien V-FITC and PI staining, respectively. The different labeling patterns in this assay identify different cell populations, e.g. region A: necrotic cells% (PI-positive/annexin Vnegative);

region

B:

late

apoptosis%

(secondary

necrosis)

(PI-

positive/annexin V-positive) ; region C: vital cells% (PI-negative/ annexin V-negative); region D: apoptotic cells% (PI-negative/annexin V-positive).

Chapter Four


Table (4-1): Score range and apoptosis % in lymphocyte isolated from CLL patients by flow cytometry machine 59

(Cll patient

Necrotic cells-

Late apoptosis

Viable cell-

Apoptotic cells-D

A (%)

cells-B (%)

C (%)

(%)

No.1

0

8.06

71.2

20.0

No.2

2.1

8.9

87.8

1.1

No.3

0

0.3

58.5

41.2

No.4

0

0.2

71.2

28.6

No.5

0

0.3

86.3

13.4

No.6

0

0.4

79.9

19.7

No.7

0

0.2

73.4

26.4

No.8

0

0.2

70.6

29.2

No.9

0

0.3

67.9

31.8

No.10

0.1

1.6

88.0

10.3

No.11

0.1

1.5

93.0

5.4

No.13

0

0

88.4

11.6

No.14

0

0.2

75.4

24.4

No.15

0

0.3

76.3

23.4

No.16

0

0.3

64.9

34.7

No.17

2.7

0.2

95.5

1.5

No.18

0.3

1.1

91.6

7.1

No.19

0

0.4

94.0

5.7

No.25

0.1

0.5

82.1

17.4

No.26

0

0.5

95.3

4.2

No.27

0.1

0.3

87.6

12.0

No.28

0

0.4

75.7

24.0

No.29

0

0.1

68.8

31.1

No.34

0.1

0.9

96.6

2.5

No.35

0.2

0.9

96.5

2.4

No.36

0.2

0.8

97.2

1.8

Number)

Chapter Four


60

No.37

0.1

0.1

96.7

3.2

No.38

0.1

0.9

96.6

2.5

No.39

0.1

0.1

89.5

10.3

No.40

25.8

0

70

4.2

Table (4-2): Score range and apoptosis % in lymphocyte isolated from healthy (control) by flow cytometry machine . Control No.

Necrotic cells-A Late apoptosis (%) cells-B (%)

No.12

0

0.1

88.0

N0.20

0.3

9.4

84.8

5.5

No.21

0

0.1

90.8

9.2

No.22

0.8

26.6

65.7

7.0

No.23(un stain)

0.6

0

99.4

0

o.1

90.8

9.2

No.24(annexinonly) 0.0

Viable cellC (%)

Apoptotic cells-D (%)

11.9

No.30

0

0.2

96

3.8

No.31

0

0.9

99

0

No.32

0.1

0

99.9

0

No.33

0.1

0

99.9

0

No.41

0

0.2

96

3.8

No.42

0.1

0.1

97.1

2.7

No.43

0.3

9.4

84.8

5.5

No.44

0.6

0

99.4

0

No.45

0

0.1

88.0

11.9

Chapter Four


61

Table ( 4-3) Flow cytometer measurements of patients& control Parameters Necrotic cells (%) Late apoptosis cells (%) Viable cells (%) Apoptotic cell (%)

Mean ± SE Patients Healthy 1.07 ± 0.03 0.17 ± 0.02

Range

t-test

0 - 2.7

0.074 *

0.639 ± 0.07

0.803 ± 0.04

0 - 1.6

0.344 NS

82.88 ± 4.73

86.80 ± 3.79

58.5 - 97.2

7.844 NS

15.04 ± 1.03

4.40 ± 0.5

1.1 - 41.2

4.261 *

* (P