Sequential Treatment of Human Chronic Lymphocytic Leukemia with

Vol.

4, 445-453.

February

Sequential with

1998

Clinical

Treatment

Bryostatin

Preclinical

1 followed

Chronic

Lymphocytic

445

Research

Leukemia

by 2-Chlorodeoxyadenosine:

Studies’

Ramzi

M.

Victor

P. Almatchy,

Mohammad,2

Christian

of Human

Cancer

Khalil Nathan

P. Schultz,

Mary

Varterasian,

Division

of Hematology

Henry and

efficacy

Katato, Wall,

Kan-zhi

H. Mantsch,

Ayad

M.

Al-Katib

into

and

Oncology. Karmanos Cancer Institute, Wayne State University School of Medicine, Department of Medicine, Detroit, Michigan 48201 [R. M. M., K. K., V. P. A., N. W.,

M. V., A. M. A-K.], and Institute for Biodiagnosties, National Research Council Canada, Winnipeg, Manitoba R3B 1Y6, Canada [K-z. L., C. P. S.. H. H. M.]

ABSTRACT We have previously

reported

induces

of

differentiation

that bryostatin

chronic

1 and

2-CdA,

we used

a xenograft

a new

batch

of severe

combined

model

immunodeficient

Bryo

1 and 2-CdA at the maximum .Lg/kg i.p. and 30 mgfkg s.c., respectively) to the mice at different combinations survival

of

in

days,

the

tumor

mice.

tolerated doses (75 were administered and schedules. The

growth

inhibition

ratio,

the

tumor growth delay, and the log10 kill of the mice treated with Bryo 1 followed by 2-CdA were significantly better than the control and other groups. We conclude that the

1 (Bryo

lymphocytic

of Bryo

CLL in WSU-CLL-bearing mice with severe combined immune deficiency. s.c. tumors were developed by injection of WSU-CLL cells, and fragments were then transplanted

Liu,

1)

leukemia

in vitro to a hairy cell (HC) stage. This study tests the hypothesis that Bryo 1-differentiated CLL cells are more

sequential treatment with suIted in higher antitumor

Bryo 1 followed by 2-CdA reactivity and improved animal

survival.

(CLL)

susceptible to 2-chlorodeoxyadenosine (2-CdA) than parent CLL cells. A recently established EBV-negative CLL line (WSU-CLL) from a patient resistant to chemotherapy including fludarabine was used to test this hypothesis. Both Bryo 1 (10-1000 nM) and 2-CdA (5.6-22.4 riM) exhibited a dose-dependent growth inhibitory effect on the WSU-CLL cell line. In vitro, the sequential exposure to Bryo 1 (100 nr’i for 72 h) followed by 2-CdA (11.2 M) resulted in significantly

higher

rates

of growth

inhibition

than

either

agent

INTRODUCTION B-cell ing

for

When

therapy

recently, time,

is the most 30%

the new at this point

very

active

patients

with

patients

2-CdA

with

fludarabine-resistant

trate-resistant

incorporation

markers,

acid CD11c

and

phosphatase CD25,

reaction after

and two

72 h of culture,

important confirming

the differentiation of CLL to HC. The Fourier transformation infrared spectroscopic analysis showed that the amount of membrane lipids significantly increased in Bryo 1-treated cells compared to controls after 24 h, whereas the protein content, as well as the DNA content, decreased. This finding supports

the

change

of CLL

to HC.

To evaluate

the in vivo

Received 7/1/97; revised 10/7/97; accepted 10/27/97. costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 1 8 U.S.C. Section 1734 solely to indicate this fact. I This work was made possible by Grant 6323-97 from the Leukemia Society of America, and it was supported in part by a grant from Ortho Biotech, Inc. (Raritan, NJ).

To whom Hematology 2

requests for reprints should be addressed. at Division of and Oncology, Wayne State University School of Mcdi-

550 East Canfield, Lande Medical Research Building, Room Detroit, Ml 48201. Phone: (313)577-7919; Fax: (313)577-7925: mail: [email protected].

dine,

317, E-

into

of action

combination

regimens

and

with

antilymphoid

products,

have

been

identified

motherapeutic

agents.

protein

C activator

kinase

animal

tumors Bugula

cells

to a HC

phological markers

Institute

I , a macrocyclic that

has

subsequent

Natural

among new treatment in combination with

Bryo

changes like CD!

(5).

This

activity. and are now modalities is standard che-

laetonc

a differentiating

and

on

lc and TRAP.

differentiation the

induction

This

study

the

Products

is based of tested

and

activity

(3), is extracted and purified from the neritina (4). It has the ability to differentiate stage

in

improve

Cancer

entering clinical trials. Also, the use of biological agents

that

inactive

their

should

agents

marine

Over

treatment

little success in extending discovery of antilymphoid

of novel

from

used.

is no curative

Program, derived

more

analogue but

(2). There

mechanisms

a number

(1).

and,

and

outcome in this disease. As part of the National

CLL

The

novel

are

agents,

leukemia

CLL

therapy for CLL, and there has been the overall duration of survival. The with

steroids,

is a purine

HC

account-

countries

fludarabine, to these

alone. Changes in immunophenotype, enzymes, lipids, proteins, and the DNA of WSU-CLL cells were studied before and after Bryo 1 treatment. Bryo 1 induced a positive tar-

agents

leukemia,

in Western

agents,

analogue. resistant

are limited.

in

adult

alkylating

purinc

become

common of all cases

is required,

patients

options is

CLL3

approximately

a on

marine CLL

on

mor-

HC-associatcd the hypothesis

The abbreviations used are: CLL, chronic lymphocytie leukemia; 2-CdA. 2-ehlorodeoxyadenosine; Bryo, bryostatin: HC. hairy cell: TRAP, tartrate-resistant acid phosphatase: ET-IR, Fourier transforma3

tion infrared; SCID, severe phatase: MID, maximum sity.

combined tolerated

immunodeficient; AeP, acid phosdose; WSU. Wayne State Univer-

446

Sequential

that

Treatment

Bryo

2-CdA

1-differentiated

than

parent

of cellular

samples,

such

example,

using

patients

‘

and

cm

strate

qualitative

ratio

Recently,

‘.

from

of two

the area

1540

also

spectroscopy,

use

anec

to treatment

eluded

from

of two

Schultz

that

patients

in

from

B-CLL of normal

bands,

regions, were

(9)

using

bearing

a CLL

strated,

by using

ET-IR

of the

WSU-CLL

cells

proteins,

and

those

line

DNA

noglobulin

at I 080

and

were

cells

express

CLL observed

FT-IR

having

lished

Studies.

in our

laboratory

refractory fludarabine; The

cell

line

shows

(3)t(p

l4;p24).

and

forms

suspension

The

penicillin cclls/ml.

medium

tially

with

State

University)

or 2-CdA 5.6,

bovine

Bryo

diluent

(0.02%

in RPMI

1640

1%

CLL

The

WSU-CLL

containing

treated

Inc.,

Raritan,

riM. was

the

14

free

analysis,

To

NJ)

an

added

200,

to the

six

triangular

centration

of S

1%

as

BSA

x

Staining

l0

described

cells/tube

and into

and washed

previously

(12).

(mouse antihuman) to B cell differentiation munoglobulins were added at saturating and

incubated

incubated antimouse)

at 4#{176}C for 35 mm. for that

30

mm was

Cells

at 4#{176}C with FITC-conjugatcd.

Arizona

and

1000

nM of

of wells,

all

films

with

of each

good

against out

prepaarea

homogenous IR

spectra

used

as described

three

spectra,

a blank

a

under used to

the sampling For

samples,

earned

were

coadded

and previ-

each

from

background.

on a FTS-60

to generate

of 8 cm The

.

smoothing

‘

with

spectra

function

used

as

The

FT-IR

a single

an encoding

were

before

then

spec-

spectrum,

interval

apodized

applying

solved

in acetone

p.g/ml

and was

previously 106

to

again cells

light. the

and

town, were

for band

of 1 with

the Fourier

Cells and

a

trans-

24-h

ceived

l0

each

were

sacrificed

pelleted, washed

Bryo

flank

were

When

the tumors

light

dis-

of 200 as described

solution

were

supematant

was

with

Samples

cells

generated

PBS. were

added

removed,

analyzed

from by flow

by combining

Four-week from

mice were as described

area.

to a concentration from

D

was

forward

fluorescence.

obtained

WSU-CLL and

the twice

1-treated

7AAD

CA)

1 ml of PBS, and mixed well. for 20 mm on ice while protected

Xenografts.

were

NY). The developed

(goat

in PBS

Actinomycin Jolla,

100 .sl of 7AAD

Scattergrams

mice

La

diluted

were was

with

half-widths

Cytometry.

at -20#{176}C,protected

Briefly,

pellet

scatter

SCID im-

Flow

suspended in cells were stained

WSU-CLL

with

mixed,

antibody

light

‘

and

and kept

(15). cells,

WSU-CLL

and

16 cm

Staining

Calbiochem-Novabiochem,

cytometry.

antibodies or

of I .6 and

7AAD

to

at a con-

antigens

Finally,

were

quality within

h

IR-transparent

disc

(7AAD,

Cytometry.

washed

on

or sequen-

concentrations, were

of the

(14).

in 3 ml of PBS

a second

24

pi

Five

(k value)

tubes

Monoclonal

WSUafter

of2 X l0 105/ml in

X

and

Flow

75-mm

(13).

of Fourier parameters

untreated aliquoted

with

Cells.

circular

was

obtained

resolution

cell

times. were

ace-

for AcP

Flemans

NaCl.

The

method

for

were

interferograms

three

to be stained

Inc.,

in 60%

cultures

chemicals.

same

Briefly,

film,

at nominal

from

Immunofluorescence

and

produced

contaminating the

10).

films.

methodology

measurements

control. Cells were counted daily for S days using trypan blue dye and hemocytometer. The entire experiment was repeated

Cells

of the

prethe

formation. In addition, band narrowing by the method self-deconvolution was performed with smoothing

number medium

of

wavenumber/cm

at concentrations

equal

(9,

stained

deposited

as thin

by IR microscopy

This

cluster

fixed

1-treated

were

down

were using

Instruments,

were

in 0.9%

as a

mm. The films were kept in a desiccator for 3-5 mm. The BaF,-window was

humidity

mm2.

smears cultures

of WSU-CLL

Bryo

106 cells) dried

checked

cul-

unit/ml

by G. R. Pettit,

of 10. 100,

1;

10% heat100

concurrently

X and

was

markers.

to Hayhoc

resuspended

(-3

films,

IR

with

in liquid

and

of 2-3 vacuum

ration

expressed

and then

and

immu-

phycoerythnin

were

Southern

Analysis

Mouse

or

or double

smears

according

control

with mono-

trometer (Bio-Rad, Cambridge, MA). equipped with a nitrogencooled mercury cadmium telluride detector. Two hundred fifty-

t(5; 12)(q3

L-glutaminc,

provided

22.4

DMSO)

with

1 3),

It grows mice.

were

washed

Results

3% gluteraldhyde

from

a different

estab-

aberrations

1 ;q22;p

in SCID

at concentrations and

was

male

chromosomal

1 (generously

16.8,

line

streptomycin at a density at a concentration of 2

and

(OrthoBiotcch,

1 1 .2,

cell

a 66-year-old

serum,

were

ously

t(4; 12; 12)(q3

G. and 200 pg/ml Cells were seeded

culture

or to

vincristinc, and prednisonc; Adriamycin, and dexamethasone.

tumors

cells

A

incubated

FITC

for single

air-dried

tartrate,

CLL

of

WSU-CLL

multiple

without

stabilize

in lipids,

leukemia.

from

maintained

fetal

demon-

to 2-CdA

and

diameter a mild

treatment

changes

sensitive

t(l8;21)(q12;p12).

s.c.

was

inactivated

the

of HC

cells

containing

were

in the dark.

with

(Shandon,

The

BaF,-windows

model

also

PA).

suspensions

METHODS

add(16)(q24)x2,

tune

I induces

with

Bryo 1. 1 followed

the in vitro

II centrifuge

tone

cells

Studies. Cytocentrifuge control and Bryo 1-treated

IR Spectroscopic

present spectra

xenograft

1 1). We

that

Bryo

to cyclophosphamide, and vincnistine,

45,x,del p13),

with

AND

Vitro

Ref.

spectroscopy,

the features

MATERIALS In

(WSU-CLL;

mouse

con-

spectra

cells. In the in the ET-IR

Cytospin

and

were analyzed. The results expressing immunofluores-

control.

positive

in PBS Dickinson).

phycoerythrin-conjugated

conjugated

of cells

I % BSA (Beckon

staining. and

as a negative

Sewickley,

sensitivity/resist-

color

of

at 4#{176}C for 30 mm

Enzymatic from the

pared

ml

cytometer

per test of cells

FITC-

isotypes

used

proteins,

and

in 0.5 flow

dual of

percentage

to demon-

(10)

the

antibodies

530

in DNA,

chlorambucil

a SCID

consistent

elonal

and

able

ex viva

For

combinations

peripheral

at 966

resuspended

on a FACScan

cence.

For

cells

the

CLL

B-CLL

and

minimum of 20,000 cells were given as a percentage

biological

of WSU-CLL cells before and after treatment with We report here the preclinical activity of Bryo 2-CdA

washed

the

normal lymphocytes. The in the FT-IR spectra of

and

2-CdA-resistant

for

purified

differences

2-CdA

tool

complex

spectral

differently from 2-CdA-scnsitive study. we examined the variation

by

to

(6-8).

et al.

to predict

with

for

those

and quantitative

cells

susceptible

or cell cultures

IR marker

and lipids between CLL cells and same group also used the differences CLL

more

a powerful

tissues,

be distinguished

and/or

,

become

fluids,

ET-IR

are

analyzed

components

by making

cm

cells

cells. has

as body

can

blood

CLL

CLL

spectroscopy

ET-IR analysis

of CLL

cells s.c.

Taconie

old

female

Laboratory

adapted and previously

WSU-CLL ( 1 1 ). Each

(in serum-free

RPMI

tumors

were

developed,

dissected

and

xenografts mouse re1640)

the

ICR-

(German-

mice

mechanically

s.c.

in

were dis-

Clinical

Cancer

447

Research

i E

z

C

C-)

C,

I

Days Effect of various concentrations the WSU-CLL cell line. E, control; Fig.

.,

of Bryo 1 on the cell growth #{149} , 10 nM; V. 100 nM; A, 200

I

1000

I

in Culture nM;

Fig.

2

into

single-cell

by

Ficoll-Hypaque

separated

suspensions.

twice

with

RPMI

typic

and

karyotypic

lished

cell line to ensure

by cutting

the tumors

planted

s.c.

group

of mice.

s.c.

Design.

groups

different

origin

bilaterally

into

naive,

S.

mice

observed

monitoring

changes

drugs.

Animals

reached

2000

for s.c. tumor

occurred,

Assessment

tumors

euthanized

when

their

of body

of

Tumor

assessing

antitumor

activity

used

in our laboratories

weight

length

and

bition when

(T/C)

(mg)

the median

T is the

median

tumor 900

median

tumor

(in

days)

tumor

required

plus

of the burden

discomfort.

end

points

group

for

the

group

inhi(7)

C, where

-

treatment

group

tumors to reach 900 mg, the control group tumors

and C is the median to reach the same

cell kill doubling

T - C/3.32 (Td), where Td is tumor involving mice were performed under

gross time.

Institutional ysis

on 48

(log10) All studies Review

animals

Board-approved with

bilateral

protocol. tumors,

using

time

(in

weight;

days)

(d)

Statistical a t test

1 and showed

with

Bryo compared

1 showed

nM,

2-CdA

on Cell

growth

At a 10 nM any significant

cells,

except

some growth either control or

compared

on

day

4.

significant

A

on day

with

the control

nrvi concentration over

with

inhibition

cells treated respectively, was

inhibition

4 (Fig.

1). At 100

and

100

dose,

nM

for

tumor

(Fig.

with

2). Isobologram

between of Bryo

Bryo 1 (100

1 1 .2 p.M of 2-CdA of 2-CdA,

1 and nM)

on day

To examine effect

respectively,

Bryo

1 showed

nrsi concentration

compared

growth

at 200 nsi concentra63 and 39% inhibiof

the 200

4) when

the

no

for growth a significant

control

cells

and

with 5.6 p.M. and exhibited 57 and 21 .3% inhibition, on day 4. 2-CdA at concentrations of 16.8 and 22.4

toxic

interaction

when

Bryo

growth

inhibition (on day 10 nM. A significant

1000

advantage

Both inhibitory

concentration,

inhibition of WSU-CLL cells was noticed tion. Bryo 1 at this concentration exhibited tion

Growth.

a dose-dependent

untreated

with

bination (a)

(C) reached T

of Bryo 2-CdA

compared

JIM

for

standard

growth

treated

=

all

Studies

I and

B are the tumor

(b) tumor

delay

from

of control.

growth

with

growth

weight

that

with

as follows:

in the control

(c) tumor

with

along

total

in the

on tumor

compared

inhibition and may not be clinically achievable. On the other hand, 2-CdA at 1 1 .2 p.M showed

(1 2) and were

weight

on the cell growth of 1 1 .2 p.M: #{149} , I 6.8

,

once

effects

A and

riM;

and

side

The

where

to

PBS

in accordance

respectively;

weight

mg;

time

B2)/2,

X

(in mm),

is the

approximately

(A

=

width

were

assigned

to avoid

Response.

procedures tumor

weight)

conducted

and

effect on WSU-CLL cells. 1-treated cells did not show

conditioned

measured

and

was

groups

Vitro

Bnyo

were

development,

weight

10%

trans-

given i.p. and 2-CdA indicated in Table 2.

were

intervals,

Effect

drug-efficacy

received

in animal

were mg (-

were

xenografts

randomly

The

of 2-CdA

A, 5.6

p.M.

experimental

In

After

of a second

similarly

were

22.4

confidence

accomplished

flanks

(animals

concentrations

cell line. N, control;

RESULTS

estab-

Mice were checked three times Once palpable tumors devel-

or as control

development

washed

the

that

the

as a diluent). Bryo 1 was according to the schedule

tumor

was

WSU-CLL

into

of 6 or more

were

with

fragments

the

then

and its stability.

0.02% DMSO was given s.c. mice

of various

to pheno-

For the subsequent

from

treatments

and

comparison

trocar

were

subjected

propagation

using a 1 2 gauge trocar. for tumor development.

oped,

were

20-30-mg

a 1 2 gauge

fragments

transplanted animals a week

into

Trial

small

for

serial

cells

centrifugation

cells

the human

tumors,

via

Efficacy

These

analysis

of s.c.

Tumor

density

1640.

formation

trials,

Effect

the WSU-CLL JiM;

sociated

4

Days in Culture

of

nM.

3

2

whether Bryo

analysis 2-CdA

with

concentrations

4 was

synergistic

Bryo

1 can

1 at 100 nsi and

to characterize

revealed

that

the

the

of either

(data

augment 2-CdA

5.6

not the

at 11.2

comor

shown). inhibitory p.M

were

chosen for the subsequent combination treatments

experiments. Of all of the different tested, only the sequential addition of

100

(72 h later)

in

nM

Bryo

complete

1 followed growth

Adding

2-CdA

growth

inhibition

inhibition

24 or 48

h after

compared

cells

95%

2-CdA

as compared (data

not

with shown).

the

i.M

WSU-CLL

Bryo

with

sequential additions (2-CdA, points showed a lesser growth

anal-

by 1 1 .2

of

1 showed

its addition

then Bryo inhibiting

concurrent

2-CdA cells

less

resulted (Fig.

3).

dramatic

at 72 h. Reverse

1 ) at different time effect on WSU-CLL addition

of Bryo

I and

448

Sequential

Treatment

of CLL

Table

Bryo

I

I-induced

enzymatic cells in vitro

changes

in WSU-CLL

w,e

Bryostatin-treated cells

I-

‘C

Stain

E

Untreated

z C,

3

strong

positivity.

2CdA

induce

CD11c

1

(22.4%)

CD1

lc,

top

panel)

(Fig.

2CdA

cells

B cell

Bryo

1-treated also

lymphoma

typically

cells

this

4

2

3

Days

in Culture

range

ence

3

Sequential addition of 2-CdA (A. 24 h; LI. 48 h; , 72 h) to 1-treated WSU-CLL cells in vitro (100 nsi Bryo 1, 11.2 p.M 2-CdA) compared with adding both agents at time zero (Bryo 1 +2#{149}).

methylene

untreated

and

Bryo

1-treated

reactions.

TRAP

AeP

and TRAP

The

reaction

specific

(positive was

for

WSU-CLL

At 72 and

HC

120 h, Bryo

reaction)

stronger leukemia

after (16,

We

in lipid,

protein,

1-treated

and

120 17)

AcP

peaks

cells

for able

to induce

cells

(Table

1).

reaction

is

acids

in

features protein

h. The

and

TRAP

is typically

negative

Effect

of Bryo

ers (CD1O, CD! le, 1-treated WSU-CLL were (2.8%),

1 on Immunophenotypic

the expression

highly CD25

positive ( 1 .3%),

of selected

CD22, cells

and at 24,

for CD1O and

CD22

Changes.

immunophcnotypic

CD25) 72,

(99%),

on

and

120

and

( 1 1 .2%).

We

and

Bryo

lipid

1 was

able

in A

found

in WSU-

with

untreated

(bottom

panel)

(RCH3),

methylenc

the treated

cells,

contents.

The

protein

5 demonstrates and

cells

here ‘

and

fewer

the existproteins

indicated cm

) and

an ester

(open

by two

positive

negative

methyl

on band

decreased

Bryo

intensity

of typical

to

CLL

cells

in Fig. 6 shows

24-h

Bryo in the infor-

band

at

1467

the

fatty

at 1740

cm

coming

from

lipids.

Typical

protein

amide

II and

I

indicate a relatively lower panel indicate an increased

content.

in DNA

the

Bryo

the

methylene

6)

panel,

represented

Spectroscopically,

Content.

lc

24-h bottom

the

is then

a different

protein

decrease

6,

between

which

1650 cm in the upper

DNA

between Fig.

difference

membrane

with

content cells.

of the top panel.

at 1550 and content. Arrows

treated

DNA

control cells,

is based

in simple

and

now

untreated

and valleys

significant were

cells

for CD!

and untreated

Decreased

Bryo

h. Untreated

negative

cm

mark-

untreated

5

For in

and 2923

spectroscopic

and

mation

CLL.

examined

the

1-treated

1 was

in WSU-CLL

was

methyl groups].

arrows)

(at 2853

Observed

and Bryo 1-treated cells value is typical for C-H

in Fig.

1-treated

of

expres-

Spectroscopy.

Fig.

change

panel)

13%

amide B bands (at 2953 and 3060 cm The spectral range between 800 and 1800 cm ‘ (Fig. provides absorbance readings which further indicate differences

examined and

bands

hand,

(2 1-23).

of lipids

as

of Bryo and mono-

a specific

Contents

of control cm ‘; this

(filled

Bryo

on

and

depicts TRAP.

lipids

in the

on

other

by FT-IR

(R3 CH)

(top

CD1O/CD2S

1 for 24 h compared

a massive

spectra

of more

leukemia

replications.

[such

methinc

indicates

difference

5

Bryo

and

Bryo

4,

CD22/CD25

on 22.4% leukemia

the

Protein Cells

three

vibrations

arrows)

AcP

with

in all

and

(R2CH,),

On

in the amount

treated cells

stretching

1 on

and

WSU-CLL

increase

and

CD22/CD25,

in HC

(Fig.

respectively, at 72 h. 200 nM of Bryo 1 for

CD22/CD1 lc is unique to HC

shows the two mean spectra in the range of 2800-3150

V

Bryo

seen

it

CD22/

cultures

of the cells,

(18-20).

in Lipid

1-treated

control

and 1.1%, cells with

coexpressed

and

4).

CD1O/CD2S,

control

le on 22.4%,

of

cells

expression,

le,

lc on 22%

coexpression WSU-CLL

cytoid

CLL

C,

of

+++,

at 72 h (Fig.

untreated

CD22/CDI panel).

significant

4

in

4, bottom

Changes

Effect

h

+++

moderate;

++,

(13%)

(92.4%)

of CD1O/CD1

CD1O/CD1

13%

The 1-treated

K

CD25

was 5, 1.3, 2.8, of the WSU-CLL

of the cells,

Bryo

and

CD22/CD25

13%

sion

CdA:

mild:

+,

expression

coexpression and

72 h induced

0 C,

Fig.

+++

++

equivocal;

-/+,

CD22

Treatment

E z

120

++

4

The

z

72 h

-

negative;

up-regulated

K

cells

-/+“

a

.

WSU-CLL

AeP TRAP

Our results content

1 for 24 h. This DNA bands

bands.

when

demonstrated WSU-CLL

can be seen The

at 1000,

IR spectra 1100,

and

a cells

by following of WSU1713

cm

Clinical

-i: 4Ti

LcD10/CO5

[oiOfCO11C

h

:

00 iQ FL1-HCOiO

-

102 FITC-’

1--i ?1

#{149}

iOl

98Z

ji 102 I gO FL1-H’CD1OFuTC-)

1O

.

FL1-H\CDiO

I 00 10’ FI.1-H\C022

10 4

103

100

FITC-

1 00

-i

22.4%

3

i

iO

10’

iO3

1o3

l:C027/C1J25

2E

in

78 -j

Differences

between

revealed

1-treated

Bryo

cells

at all

Induction

of

these

fluorescence-activated

trol and panels).

Bryo

1-treated

This

shows

cells),

and

cells (R3-live

from

8%

cells),

7AAD-dim

(Ri-late

+

8%

in

in Fig. defined

of con-

7AAD

control

to

24%

io

FI’TC

or the concurrent

not show

any

and control

cycles

(X2)

tumors Mean

Studies 2 shows and their

1, 2-CdA,

Bryo

1 was given

after

2-CdA.

(12),

and

Bryo

1 was was

in this

the MTD

the basis

their

3 shows

against

is illustrated a TIC

Using

Bryo

antitumor

activity,

after

Bryo

for Bryo

used

MTD, When

at its MTD

2-CdA

was

Bryo

was

group.

On

1 . This

=

(%),

T

-

a drug

1 alone 0.90)

2-CdA however, was

true

0.05,

1, 2-CdA, mice

C (days), is considered

was

against showed

active

log10 active

(T/C

=

19%;

WSU-CLL-SCID no activity. The

was

seen

when

for

both

the

2-CdA one

and

of tumor

0.03,

and 0.03,

differences (alone),

were

the group

We have

one

indicate 1 ) or two

(X

the duration other

weight

in mg,

showed

that

for the

treatments

(Fig.

as Ps,

as well

there

respectively. observed

were

were

significant

given

followed

Bryo

by 2-CdA

On the other

hand,

between

the group

1 + 2CdA,

the group

for 5 days,

previously

of CLLs

in vitro

reported

that

to a HC

stage

Bryo (5).

and

hypothesis that Bryo 1-differentiated CLL ceptible to 2-CdA, a drug active in treating

kill.

mia

if it

recently a patient

xenografts.

and

in extending with

The in all

the results

1 for either

compared

3). Results

1 for 3 days

tiation of Bryo

mg.

S days

xcnografts

Clearly,

Bryo

for activity.

and

no

given given control

DISCUSSION

of 30 mg/kg/day

2).

activity

studied.

after

2-CdA

antitumor

did

Admin-

p.glkg/

35 mg/kg/day.

a dose

were

were

addition agents,

mice.

SCID

16 and

between the control group and the groups with Bryo or Bryo I followed by 2-CdA (either X 1 or X2). The

significant

which 1 (75

(Table

differences 1 (alone) PS

or

2-CdA

Bryo

remained

Briefly,

and before

42%.

log10 kill whereas

=

was its

with

we used

criterion,

13; xenografts, C

at

WSU-CLL-SCID

by TIC

response

achieves TIC Accordingly,

in mice

antitumor

combinations

Activity

i.p. and s.c.

of 2-CdA, (Table

design

2-CdA,

to be 35 mg/kg/day.

for 2-CdA studies

Table

injected

or sequentially

of the MTD

in subsequent

with

injected

study

trial

in one and two cycles.

concurrently

in combination

day),

preclinical

combinations alone,

2-CdA

determined given

the efficacy

WSU-CLL

effective

2000 range

sequential

by any

I 1%,

20 days,

use of both

followed

of

were

12%,

reverse

did not show

was more

and

values

3 days

given

to reach

The

or with 200 nsi In each reading. the

top)

and

WSU-CLL-bearing

weight

2-CdA

that

kill

against

later)

again

calculated Table

1 for

treatments

8).

Vivo

activity

tumor

24 h.

In

2-CdA,

median

after

log10 schedule

schedule.

1 and

4 days

16%

C, and

-

1 .36 for the two-cycle

Bryo

As

T

of Bryo

(starting

cells).

+

TIC,

1.10 for the one-cycle

istering

to induce apoptosis on 16% of 1 increased late apoptotic +

the

The

and

right

(R2-apoptotic or dead

days,

stain-

and

7 (left by

apoptotic

in Fig. 7, Bryo 1 was able WSU-CLL cells at 24 h. Bryo

by Bryo.

scattergrams

regions

shown

apoptotic

Cells

sorting

arc shown

three

7AAD-bright

schedules.

WSU-CLL

of the 24 h Bryo

in WSU-CLL cell

the

untreated

content

bands.

of Apoptosis

7AAD-negative

and

in the DNA

Typical

ing:

1-treated

a decrease

8Z

-

FL1 i4CO22

Fig. 4 Two-color flow eytometrie analysis of WSU-CLL cells in vitro at 72 h. WSU-CLL cells were cultured alone (control: Bryo I (bottom) for 72 h at 37#{176}C. Panels from left to right show CDIO/CDI Ic, CDIO/CD25. CD22/CDI le, and CD22/CD25. number of cells gated was kept constant.

cells

i04

‘

137.

_FL1.H’CO22_FUC-

FITC-

ioz FITC

?!.

71Z iO

‘

iOi HCD22

FLI

!

102

ji

FL1-H\colo

A

in

lOt

FITC-’

1:C022[CIJ11C

-

87Z -

:

8Z

104

22%

I ,

‘.

13%

478Z

10r-ro

i3

.

l:COlO/C025

CiflO/COh1C

!:

:

t:

I

2.8%

1.3%

::

....

,%

,-

J

5Z

449

Research

CON 0I.CO22fCt

)L.C022/COI1C

A

A5

Cancer

T

-

mice highest

used

but

not

active

established resistant

to test

in fludarabine-resistant EBV-negativc to chemotherapy

this

1 induces This

hypothesis.

cells de

are nova

given

2-CdA

two

cycle

This

after sequential

Bryo

I resulted

interaction

Bryo

A from was

for the first CLL tumors administering

antitumor 1 and

the

susleuke-

(24-26).

we demonstrate

in improved between

more

(WSU-CLL) fludarabine

time that Bryo 1 given to SCID mice bearing showed reasonable antitumor activity. Moreover,

was

differentested HC

CLL

CLL line including

Here,

study

2-CdA

activity. is se-

450

Sequential

Treatment

of CLL

1

Lipids

1 Difference Bryostatin

-

control

LII

LtI Protein

Fig. 5 The difference in spectra demonstrates the existence of more lipids (filled arrows) and fewer proteins (open arrows) in the Bryo 1-treated cells. indicated by two positive methylene bands (at 2853 and 2923 cm ‘ ) and negative methyl and amide B bands (at 2953 and 3060 em ‘).

C (U

-e 0 U)

.0

Control

----

Cells

.

1_

.

I

I

.

I

2850

2800

2900

.

.

I

2950

quence

dependent

quential Bryo years

because

use of the two 1 has

neither

agents

received

mon activity,

Bryo

entiating

indicate

growth

inhibitory

on

that

Bryo

1 ( 100 nM) followed complete

(Fig.

in the HC stage Bryo

reaction)

in WSU-CLL

specific

2-CdA

powerful

show

inhibition

past

1 was

for HC Bryo

In

differ-

(3).

our

vitro,

1 and

by 2-CdA of

2). The

I also

usually

able

leukemia induced

express

to induce

cells (16,

(Table

specific

cells

AcP

and

I ). The

17) and

the expression

enzymes TRAP

TRAP

is typically of specific

Subsequently,

lines

(20)

(positive

and

FT-IR of

it is

and

lipids)

Drcxlcr

able

to

features

the peripheral

of

blood

et al. (3)

reported

that

of

B-CLL

cells

differentiation

has

and

DNA

peripheral

negative

in

this

technique

has

of CLL of

by studying

CLL

treatment ehlorambucil resulted

cells with

to

to

of

normal

higher

(18, Bryo

phenotypic

from

demonstrate

that

non-Hodgkin’s

lymphoblastic

used

with

normal

which is also typically seen in HC leukemia is consistent with our previous findings that

(5).

were

and

taken

inducing

comparison than

CD22/CD25,

of

spectroscopy CLL;

have

imporle and

disease

acute

is

phenotypic

cells

of the human

reaction

4). More CD22/CD1

CLL

we

modulation

nosis

and

such as CD! le and CD25 1-treated cells eoexpressed

This

(Fig.

enzymatic,

on fresh

Bryo

1

B-lymphoma

leukemia

cell

a powerful

tool

line

Rch

(27).

( 1 1 .2 p.M)

WSU-CLL

morphological.

leukemia

induces cell

a dose-dependent

(Figs.

markers, tantly, Bryo HC

20-23).

1 induces

of patients with this Bryo 1 was capable

few

to its antitu-

27).

cells

3150

proteins.

3). Cells

markers.

CLL.

in the

(20,

72 h later

growth

se-

HC

to possess

on WSU-CLL

resulted

reverse

In addition

tumors

1 and

effect

use of Bryo in

found

B-cell

nor

attention

(3-5).

I has been

properties

results

agent

I

3100

activity.

considerable

as an antincoplastic

.

3050

I cm1

concurrent

showed

Bryostatin

+

I

3000

Wavenumber

cells

cellular

purified

CLL

protein blood

been

extended

the

lipid

observed ex

viva

antileukemic show that

content

and

CLL cells.

to evaluation

(DNA,

from

patients cells

and a lower

mononuclear

the variations predict

(9).

content

in diag-

components

cells

lymphocytes

two common (10). Here we

in a higher

become study

lipid More

content recently,

of the

prognosis

in the ET-IR sensitivity/resistance

agents, treatment lower

protein

in

usually

spectra to

2-CdA and with Bryo 1 and

DNA

Clinical

Cancer

Research

Lipid

1

Lipid

4, i;en;

f t

Bryostatin

-

t

controlp

DNA

Protein DNA

Fig.

6

IR spectra

of untreated

and

Bryo

‘I)

1-treated

U C

WSU-CLL cells shows bands specific for DNA at 1000. 1 100, and 1713 em’ (botto,n panel). These

rotein3l

(U .0

bands showed reduced intensity after 24 h of Bryo I treatment (data represent average of three experiments). This reduced intensity is the difference between Bryo I -treated and control cells (top panel).

I.-.

0

U)

.0

Cells

+

Bryostatin

Control 800

1000

cells

1200

1400

Wavenumber

:.:

I

. .

‘

I.

8%R1

.

24%R1 Seattergrams of 7AAD-stained cells are Left, untreated WSU-CLL cells: rig/it, WSU-CLL cells treated with 200 nM concentration of Bryo 1 for 24 h. RI, late apoptotie or dead Fig. 7 shown.

I.

8%R21

16%R21 ‘

84%R3

:-

‘

-

iOO 6OiJ’ iO#{243} iObO

‘

FSC-H\FSC-Height---)

cells,

which

structure

and/or

may explain

‘2O#{244}

cells;

R2,

forward

60%R3

.

apoptotie light scatter;

cells; FL?.

R3,

live

7AAD

cells;

FSC,

fluorescence.

4O0 6O0 aOO

FSC-H\FSC-Height---

(Figs. S and 6). Because these findings suggest

membrane

1 cm

.

.

contents membrane,

1800

1600

lipids that

in the whole the morphological

arc located there are composition changes

in the cell changes in

size)

of the CLL

ation

of membrane

larger

anee

of CLL

(e.g.,

treated

and

cytoplasmic

with

Bryo

extensions

1 . Previously, lipids

to chlorambucil

plays

our

seen

in

group

found

an important and

WSU-CLL role

to the nucleoside

that

cells the alter-

in the

resist-

analogues

451

452

Sequential

Treatment

of CLL

Table

-

2

in

Bryo

vito

1 and

Agent”

do se and

2-CdA

Dose”

Diluent (control) Bryostatin 1 2-CdA Bryo I + 2-CdA (2 cycles) Bryo-2-CdA (I cycle) Bryo-*2-CdA (2 cycles) 2-CdA--*Bryo (2 cycles) Bryo-2-CdA (3 + 5)’

0.0 75 30 75 30

schedule

for

W SU-CLL-bearing No.

Route

mgfkg/inj/day’ p.g/kg/inj/day mg/kg/mi/day

p.g/kg/inj/day mg/kg/mi/day

SCID

of animals

Table

Diluent

Bryo

In vito

3

T/C (%)

20 10 10

i.p.

7

20

6 6 6 6

10 20 20 8

s.c. i.p.-s.c. i.p.-s.c. s.e.-i.p. i.p_-+s_e_

(control)

100

I

activity

T

of Bryo

19

( 1 X)

Bryo-2CdA 2CdA-’Bryo Bryo-2CdA

(2X) (2X) (3 + 5)”

Significant at P = 0.05. NS, not significant at P 0.05. ‘. Highly significant at P = 0.05. ‘I Three days of Bryo I , followed

against

Log10

for 2 weeks a week.

(gross)

Mean

0.00

7 16 20 4 0.00

agent

followed

(mg)

by second

Range

1557.0 400.7#{176} 955.8 NS” 586.5 NS 260.0’ 243.3’ 509.2NS 1 1 19.4 NS

0.90 0.00 0.48 1. 10 1.36 0.27 0.00

0.00

(first

model

WSU-CLL-SCID

kill

13

60 I1 12 67 88

(2X)

Bryo-*2CdA

2-CdA

0.00

100

2-CdA Bryo + 2CdA

1 and

C (days)

-

of injections

6” 6 6

1 cycle (for one agent), treatment for 2 weeks, 5 days a week. I cycle (for two agents). treatment 5 days a week. 2 cycles, treatment for 4 weeks (first agent followed by second agent), 5 days I’ Doses were determined based on previous experiments with these drugs. ‘ mi, injection. ‘I SCID mice with bilateral tumors. ‘, Three days Bryo 1, followed by 2-CdA 4 days later.

Agent

No.

i_p. i_p. s.c.

“

agent).

mice

(3376-836 mg) (1626-14 mg) (1436-140 mg) (1372-14 mg) (800-14 mg) (726-14 mg) (1099-14mg) (2745-14 mg)

“

I’

by 2-CdA

4 days

later.

or simply Bnyo

C)

E

flow

C

within

showed

cytometric 72

cellular

DNA

content)

unique

and

Bryo

1 can

active 5

101520253035404550556065

2-CdA

Days Fig. 8 various 2-CdA

SCID xenografts in (X2); LL Bryo+ (X2).

HC

‘

and in

the

1-treated can

be

an

1 treatment.

(10).

spectra

cells.

or even

2-CdA

To

indicate

A possible

enhanced

induction

The a

lower

explanation

metabolism

of programmed determine

bands

whether

at 1000, DNA

1 100, content

for this reduction

(increase

in vesiculuar

cell death the

cells

subsequent underwent

and in

1713

one

Bryo

tical

of DNA

that

activity

cycle analysis

and

apoptosis

compared

12%, revealed

0.03)

cycles,

shows

the

other

treatment that

were and

there

was

tumors the

Bryo

significant

2-CdA ratio

kill

16 days, two

highest

1 and

inhibition the log10

2).

but not

WSU-CLL

after

a

1 and

(Table

showed

11%,

1.36

Bryo

to 2-CdA

growth C) and

is very

(T/

in Bryo 1.10

after

cycles.

Statis-

tumor

weight

1 was given alone (P = 0.05) and when by 2-CdA for one (P 0.03) and two (P =

compared median

with

is

CLL.

in vivo,

conducted

bearing 2-CdA

-

which using

were

and

to treat

2-CdA,

by

(T

20 days,

strategies

resistant

change

to 2-CdA,

combinations

tumor

to protein

hypothesis

mice

to HC stage

and

this

was

SCID

delay

bodies)

new

model

1 followed

cells

sensitive

with

WSU-CLL

growth

to Bryo

for

and

3). The

by 2-CdA and

with

decrease

in CLL

To test

(Table

decrease when Bryo Bryo 1 was followed

apoptosis

basis

and

in conjunction

Bryo

the tumor

seen

leukemia.

1 . Interestingly,

1 followed fludarabine

the

the WSU-CLL

showed

antitumor C),

form

be used

with

treated staining

1 induced CLL

lipid

to that

against

treated

Bryo

phenotypic),

(increase

similar could

and

schedules

to Bnyo

that

were

of 7AAD

1 to differentiate

of different

Results

Median tumor weight in mg of WSU-CLL treatments. 0, control: A, 2-CdA--Bryo (X2): A. Bryo-2-CdA (X I); S. Bryo-2-CdA

contents

combinations

cm

of Bryo

against

series

indicate

enzymatic,

basic

cells

results

7).

ability

(morphological,

II

WSU-CLL

72 h. The

analysis

h (Fig.

The C)

necrosis,

1 for 24, 48, and

with tumor

the untreated weight

of

control

WSU-CLL

group. in

SCID

Fig.

7 xc-

Clinical

nografts.

Conversely,

administering

concurrently

with

worth

that administering

by

noting 2-CdA

activity

starting (Table

finding: the

tibility that

Bryo later,

1 therapy (this

tumors

did

to grow

immediately

hiatus

was

following

based

at the beginning It had were

been

hiatus

between

the 4-day

of the 2-CdA);

suggests

2-CdA

Bryo

has

I for full benefit.

convenience;

drugs

14.

the

deoxycytidine 5’-nucleotidasc

levels

of patients who (28). The levels

responded of these

lower in CLL in nonresponders

1-treated WSU-CLL results showed that

5’-nucleotidase

ratio

(mean

Bryo

cells

at

I -treated

cells

In summary,

of three

replications)

was

with

and

in vitro

in vivo

were to two

of Bryo

1 and 2-CdA

sequential

carefully

clinical

trials

CLL.

against

in

(data

studies

age the evaluation designed

levels

higher

controls

not

encour-

regimens

in

54:

H.

resolution

H.

New

enhanced

1992.

insight

infrared

into

spectra.

protein Bioehim.

952:

115-130,

1988.

1996.

2244-2251,

87:

simplicity of use other techniques.

16. Traweek, S. T., Sheibani, K., Winberg, C. D., Mena, R. R., Wu, A. M., and Rappaport, H. Monocytoid B cell lymphoma: its evaluation and

relationship

to other

low-grade

B-cell

neoplasms.

Blood,

73: 573-

578, 1989. 17. Yam, isoenzyme

L., Li, C., and Lam, K. Tartrate-resistant acid phosphatase in the reticulum cells of leukemie reticuloendotheliosis.

N. EngI. J. Med., 18.

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