Mol Biol Rep (2013) 40:3043–3048 DOI 10.1007/s11033-012-2378-1
Leukemogenesis as a new approach to investigate the correlation between up regulated gene 4/upregulator of cell proliferation (URG4/URGCP) and signal transduction genes in leukemia Yavuz Dodurga • Yes¸ im Oymak • Cumhur Gu¨ndu¨z • N. Lale Satıroglu-Tufan • Canan Vergin • Nazan C ¸ etingu¨l C ¸ ıg˘ır Biray Avci • Nejat Topc¸uog˘lu
•
Received: 22 October 2012 / Accepted: 17 December 2012 / Published online: 25 December 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract The aim of the study is to the determine the profiles of cell cycle genes and a new candidate oncogene of URG4/URGCP which play role in leukemia, establishing the association between the early prognosis of cancer and the quantitation of genetic changes, and bringing a molecular approach to definite diagnosis. In this study, 36 newly diagnosed patients’ with ALL-AML in the range of 0–18 years and six control group patients’ bone marrow samples were included. Total RNA was isolated from samples and then complementary DNA synthesis was performed. The obtained cDNAs have been installed 96 well plates after prepared appropriate mixtures and assessed with LightCyclerÒ 480 Real-Time PCR quantitatively. CHEK1, URG4/URGCP, CCNG1, CCNC, CDC16, KRAS, CDKN2D genes in the T-ALL group; CCND2, ATM, CDK8, CHEK1, TP53, CHEK2, CCNG2, CDK4,
Y. Dodurga (&) Department of Medical Biology, School of Medicine, Pamukkale University, Kınıklı Kampu¨su¨ Morfoloji Binasi Kat:3, Kınıklı/Denizli, Turkey e-mail:
[email protected] Y. Oymak C. Vergin Department of Pediatric Hematology and Oncology, Dr. Behcet Uz Hospital, Izmir, Turkey C. Gu¨ndu¨z C¸. Biray Avci N. Topc¸uog˘lu Department of Medical Biology, School of Medicine, Ege University, Izmir, Turkey N. L. Satıroglu-Tufan Department of Medical Genetics, School of Medicine, Pamukkale University, Denizli, Turkey N. C ¸ etingu¨l Department of Pediatric Oncology, School of Medicine, Ege University, Izmir, Turkey
CDKN2A, E2F4, CCNC, KRAS genes in the precursor B-ALL group and CCND2, CDK6 genes in the AML group have shown significant increase in mRNA expression level. In the featured role of acute leukemia the regulating signaling pathways of leukemogenesis partially defined, although identification of new genetic markers in acute leukemia subgroups, will allow the development of early diagnostic and new treatment protocols. Keywords Leukemogenesis URG4/URGCP Acute leukemia Cell cycle genes
Introduction Leukemia, is the most common malignant disease of childhood and significant improvements have been achieved in its treatment in the last four decades. Five-year survival is around 80 % in acute lymphoblastic leukemia (ALL) and 40–50 % in acute myeloblastic leukemia (AML). An increasing chance of cure has been provided after determining the risk groups evaluated with cytogenetic and molecular findings in particular [1]. In addition, immunophenotyping and molecular genetic studies have improved the identified risk criteria and appropriate treatment choices increased cure rates. These success rates decreased when potentially cured childhood leukemia is repeated. Hence, identification of new molecular markers and the development of new chemotherapeutic agents are still needed [2]. The most likely mechanisms that cause leukemic transformation with clonal proliferation from multipotent hematopoietic stem cells are chromosomal translocations, loss of tumor suppressor genes, abnormal centromere duplication, dysfunction of genes that function to the sorting and separation during mitosis and activation of an oncogene that has
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not been activated before. The cell that cannot get out of the cycles after oncogene activations, which may be effective in different stages of the cell cycle, continues to grow steadily [3]. Molecules that have been studied for the development of treatment options are targeted at apoptosis or cell differentiation. For the identification of such targets, the mechanisms related to cell cycle that plays a role in leukemogenesis are needed to be known in detail. Cyclins and cyclin-dependent kinases (CDK) play a role in various stages of cell cycle control and their synthesized amounts vary in certain stages. Oncogenes are thought to play a role in leukemogenesis by affecting the control steps of cell cycles and by changing the expression levels of cyclins and CDKs [4–6]. Up regulated gene 4/Upregulator of cell proliferation (URG4/URGCP), which is located in the long arm of the chromosome 7, has been found to be associated with cellular proliferation in solid tumors, but its role in leukemogenesis is not known yet. URG4/URGCP is strongly expressed in hepatocellular carcinoma, gastric cancer and osteosarcoma. Over-expression of URG4/URGCP stimulated cyclin D1 mRNA expression, and RNAi-mediated URG4/URGCP silencing also diminished cyclin D1 mRNA expression in HepG2 cells. These results suggest that cyclin D1 up-regulation contributes importantly to the mechanism of URG4/URGCP-mediated hepatocellular growth [7]. Hence, URG4/URGCP may be a putative oncogene that contributes importantly to multistep carcinogenesis and cell cycle regulation [8–11]. The aim of this study is to analyze the functional roles of a new oncogene URG4/URGCP and certain genes involved in cell cycle at the RNA level in leukemogenesis.
Materials and methods Cases Six control groups, having no malignites, with bone marrows needed to be taken during routine screening for diagnostic purposes, and 36 cases 25 of which were ALL (15 Precursor B-ALL and 10 T-ALL) and 11 of which were AML between the ages of 0–18 applied, from March to December 2009, to the Ege University, Department of Pediatric Oncology, Dr. Behc¸et Uz Children’s Hospital Hematology and Oncology Clinic and Ege University, Department of Medical Biology, and they were taken to be studied on.
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bovine serum (FBS), 2 mm L-glutamine, 1 % penicillin/ streptomycin have been used as cell culture medium. Cells were incubated at 95 % humidity, 37 °C and with 5 % CO2 until adequate reproduction from cells has been obtained. Total RNA isolation from bone marrow samples and cell line Total RNA has been isolated from bone marrow samples and CCRF-CEM cell line (1 9 106 cells) using High Pure RNA Isolation Kit (Roche Applied Science, Mannheim, Germany). Real-time online reverse transcriptase PCR (RT-PCR) cDNA synthesis RT2 First Strand Kit (Roche Diagnostics, Germany) has been used for the synthesis of complementer DNA, from bone marrow and cell line as first step of reverse transcription. LightCyclerÒ 480 real time PCR Quantitative RNA expression analysis of 43 genes including URG4/URGCP and control genes have been designed for 96 well microplates at LightCycler 480 Real-Time (RT)-PCR platform, Names of these genes are given in Table 1. Statistical analysis The analysis of the findings has been made with the DDCT method and quantitated with a computer program named LightCyclerÒ 480 Quantification Software. The comparison of cases and controls has been performed with ‘‘Volcano Plot’’ analysis, from ‘‘RT2 ProfilesTM PCR Array Data Analysis’’, which is assessed statistically using the ‘‘Student t test’’. Moreover, parametric and non-parametric tests of cases and controls have been evaluated with the SPSS 15.0 statistical analysis program.
Results The cases have been taken under treatment as leukemia at the Department of Pediatric Oncology, Ege University Faculty of Medicine, and at Dr. Behc¸et Uz Children’s Hospital Hematology and Oncology Clinic. Characteristics of Case Groups were summarized in Table 2.
Culturing of leukemia cell line
Gene expression analysis with RT-PCR
CCRF-CEM (ALL-T cell) cell line was used for positive control. RPMI 1640 (Biological Industries), 10 % fetal
After total RNA was isolated from 36 cases and 6 control groups’ bone marrow samples, the cDNA synthesis have
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Table 1 Cell cycle control genes and URG4/URGCP Symbol
Definition
Gene name
ABL1
C-abl oncogene 1, reseptor thyrosine kinase
ABL/JTK7
ATM
Ataxia telangiectasia mutation
AT1/ATA
ATR
Ataxia telangiectasia and Rad 3 related
FRP1/MEC1
BCL2
B cell CLL/lymphoma 2
Bcl-2
CCNB1
Cyclin B1
CCNB
CCNB2
Cyclin B2
HsT17299
CCNC
Cyclin C
CycC
CCND1
Cyclin D1
BCL1/D11S287E
CCND2
Cyclin D2
KIAK0002
CCNE1
Cyclin E1
CCNE
CCNF CCNG1
Cyclin F Cyclin G1
FBX1/FBXO1 CCNG
CCNG2
Cyclin G2
Cyclin G2
CCNH
Cyclin H
CAK/p34
CDC16
Cell division cycle 16 homolog (S. cerevisiae)
APC6
CDC2
Cell division cycle 2, from G, S and from G2, M
CDC28A/CDK1 CDC20A/p55CDC
CDC20
Cell division cycle 20 homologue (S. cerevisiae)
CDC25A
Cell division cycle 25 homologue A (S. pombe)
CDC34
Cell division cycle 34 homologue (S. cerevisiae)
E2-CDC34/UBC3
CDK2
Cyclin dependent kinase 2
p33(CDK2)
CDK4
Cyclin dependent kinase 4
CMM3/PSK-J3
CDK5R1
Cyclin dependent kinase 5, regulator Sub-unit 1 (p35)
CDK5P35/CDK5R
CDK5RAP1
CDK5 regulator sub-unit related protein 1
C20orf34/C42
CDK6
Cyclin dependent kinase 6
PLSTIRE
CDK7
Cyclin dependent kinase 7
CAK1/CDKN7
CDK8
Cyclin dependent kinase 8
K35
CDKN1A CDKN1B
Cyclin dependent kinase inhibitor 1A (p21, CIP1) Cyclin dependent kinase inhibitor 1B (p27, KIP1)
CAP20/CDKN1 CDKN4/KIP1
CDKN2A
Cyclin dependent kinase inhibitor 2A (p16, CDK4 inhibitor)
ARF/CDK4I
CDKN2B
Cyclin dependent kinase inhibitor 2B (p15, CDK4 inhibitor)
CDK4I/INK4B
CDKN2C
Cyclin dependent kinase inhibitor 2C (p18, CDK4 inhibitor)
CDKN2D
Cyclin dependent kinase inhibitor 2D (p19, CDK4 inhibitor)
CDKN3
Cyclin dependent kinase inhibitor 3
CDI1/CIP2
CHEK1
CHK1 control point homologue (S. pombe)
CHK1
CHEK2
CHK2 control point homologue (S. pombe)
CDS1/CHK2
c-MYC
v-MYC myelocytomatosis viral oncogene homologue
E2F4
E2F transcription factor 4, p107/p130 attachment
HRAS
v-Ha-ras Harvey rat sarcoma viral oncogene homologue
KRAS
v-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue
NRAS
Neuroblastoma RAS viral (v-ras) oncogene homologue
RB1
Retinoblastoma 1
OSRC/RB
TP53 URG4/URGCP
Tumor protein p53 Up regulated gene 4/Upregulator of cell proliferation
LFS1/TRP53 URG4/URGCP
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase
G3PD/GAPD
ACTB
Beta Actin
PS1TP5BP1
HGDC
Human genomic DNA contamination
HIGX1A
RTC
Revers transcription control
RTC
PPC
Positive-PCR control
PPC
E2F-4
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3046 Table 2 Characteristics of case groups
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Characteristics
ALL (n = 25)
AML (n = 11)
Average
82.08
117.45
SD
49.09
69.45
Min–Max.
21–199
12–204
Male
16
8
Female
9
3
Average
66.7
53.0
SD
100.3
71.19
1.7–370 (Min–Max.)
2.3–225.58 (Min–Max.)
Precursor B-ALL
15
–
T-ALL
10
–
Age (month)
Gender
Leukocyte in Diagnosis (9103/ll)
Immunophenotyping
Risk Groups Standard risk (SR)
7
–
Medium risk (MR)
9
–
High risk (HR)
9
–
Reaction to Treatment Day 8
[1,000
19
6
Day 15
M1
M3
19
6
Day 33
Remission
No remission
23
2
been performed by using RT2 First Strand Kit (Roche Diagnostics, Germany). cDNAs have been put into 96-well plates and, in LightCyclerÒ 480 Real Time PCR, the expressions of their genes, all of which are given in Table 1, have been quantitatively evaluated in case and control groups. Analysis of gene expression changes in case groups and control groups was performed with the specific analysis ‘‘Volcano Plot’’. In T-ALL group is evaluated, the genes whose expression increases have been found significant are CHEK1 (p = 0.021), URG4/URGCP (p = 0.043), CCNG1 (p = 0.001), CCNC (p = 0.00009), CDC16 (p = 0.03), KRAS (p = 0.025), CDKN2D (p = 0.035). When precursor B-ALL group is evaluated by ‘‘Volcano plot’’, the genes whose expression increases have been found significant are CCND2 (p = 0.0044), ATM (p = 0.023), CDK8 (p = 0.044), CHEK1 (p = 0.011), TP53 (p = 0.0141), CHEK2 (p = 0.029), CCNG2 (p = 0.019), CDK4 (p = 0.012), CDKN2A (p = 0.039), E2F4 (p = 0.004), CCNC (p = 0.006), KRAS (p = 0.017). In AML group is evaluated, the genes whose expression increases have been found significant are CCND2 (p = 0.027) and CDK6 (p = 0.049). In ALL cases of all risk groups, URG4/URGCP gene expression; in higher risk group, gene URG4/URGCP 40.44
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\1,000
– – –
Table 3 URG4/URGCP gene expression changes according to risk groups Risk groups
Change
p value
Standard risk
3.28
0.008
Medium-risk
3.27
0.03
40.44
0.36
High risk
Table 4 URG4/URGCP gene expression change in two high-risk groups and a medium-risk precursor B-ALL, with t (4:11) positivity Position
Gene symbol
Change
p value
C6
URG4/URGCP
9.17
0.022
times (p = 0.36); in medium-risk group, 3.27 times (p = 0.03), and in the standard risk group, 3.28 times (p = 0.008) expression increases have been detected (Table 3). When considered in terms of URG4/URGCP gene expression, in our cases, two high-risk groups and a medium-risk precursor B-ALL, with t (4:11) positivity, 9.17 times expression increase has been detected (p = 0.022) (Table 4).
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According to the performed treatment, on the 8th day, the number of blasts in response to treatment have been [1,000, and on the 15th day of treatment response, in one precursor B-ALL group case and in five T-ALL cases, which have not entered remission, 53.29 times increased gene expressions have been observed. However, this increase could not be determined significantly (p = 0.29). In this group, in cases of one precursor B-ALL group and one T-ALL, which have not entered remission, 204.48 times increased gene expressions have been observed. This increase could not be statistically evaluated due to inadequacy of cases. One t (8:21) positive AML M2 case and two inv16 positive AML M1 and M2 cases have been evaluated according to URG4/URGCP gene expression increase, and 2.91 times expression increase have been observed. This increase could not be statistically evaluated due to inadequacy of cases.
Discussion The changes in leukemogenesis cell cycle are becoming clearer with molecular genetic research data and technological developments. This would be the starting point for the development of targeted treatments, also revealing the prognostic significance associated with the risk groups. Cyclins regulate and direct the expression of genes related to CDK complexes, CDKI genes, cell cycles, differentiation, apoptosis, DNA repair systems. As the systems which are the control points of cell cycles are responsible for the regulation and division of chromosomes and for the unity of the genome, imperfections in these systems can be the main reason of aneuploids in cancer cells and of genomic instability. URG4/URGCP, which is considered to be one of the oncogenes involved in the checkpoints of cell cycles, is located on the short arm of chromosome 7. When URG4/URGCP was first identified in 2002, that the data suggested that URG4/ URGCP might contribute to the transformation of HBx antigen-positive liver cancer cells into cancer cells in a multistep process [8]. In this study, the candidate oncogene expression of URG4/URGCP in leukemia, whose role is unknown in leukemogenesis, and the genes that might influence development of leukemia have been investigated. In T-ALL, significant increase in gene expressions of CHEK1, CCNC, CCNG1, CDC16, CDKN2D (p19), KRAS, URG4/URGCP have been determined according to the control group. In Precursor B-ALL case group, gene expression significantly increase has been observed in ATM, CHEK1 and CHEK2, which are responsible for stopping the cell cycle and are at cell cycle control point, CCNC, CCND2 and CCNG2 of
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cyclin genes, CDK4 and CDK8 of cyclin dependent kinase genes, CDKN2A (p16) of cyclin dependent kinase inhibitor genes, and also in TP53, K-RAS and E2F4. ATM gene is from the family of phosphatidyl 3 kinase/ phosphatidyl 4 kinase, and like p53, it is in close relations with important molecules responsible for important control steps in cell cycle. It is known that ATM has a role in response to DNA double-strand breaks after exposure to ionizing radiation or radiomimetic agents [12, 13]. ATM, in relation with CHEK1/2, has an important role in the control steps of cell cycle [14]. In some studies, it has been used to evaluate the reaction to the treatment of acute leukemia [15]. p53, in relation with ATM, has a protecting role in the encounter of cell with stress in G1 and G2 control points by stopping the cell cycle and inducing apoptosis [16, 17]. The fact that there has been increase in gene expressions of ATM, p53 and CHEK1/2 in pre B-ALL cases in our study may be an indicative of reaction to increased DNA damage. Despite this mechanism that tries to repair DNA, synchronization failure with proteins involved in cell cycle or, depending on another antagonizing mechanism, leukemia cells are thought to be constantly proliferate. p16 protein has been shown to inhibit progression in G1 phase [18]. However, in childhood leukemia, it is considered to be of prognostic significance, as it shows high-expression during relapses [19]. Moreover, changing expression amounts have been emphasized to be contributing to leukemogenesis with promoter methylation [20]. That the gene expression of p16 has been increased 18.86 is statistically significant in our pre B-ALL cases and it is considered to be contributing to leukemogenesis in this group. E2F4 is a transcription factor that is activated by phosphorylation of Rb protein. E2F4 activation leads to proliferation by inserting the cell in S phase [21, 22]. K-RAS mutations also contribute to leukemogenesis [23]. In our study, the expression increase of E2F4 and K-RAS in pre B-ALL group support these. There are increased or decreased expressions of cyclin genes in leukemogenesis [24–26]. Our study also proves that the increase in the expression of cyclin C, by activating E2F4, accelerates pre-B cell proliferation. It is previously suggested that URG4/URGCP is overexpressed in gastric cancer tissue compared to the adjacent gastric healthy tissue by immunohistochemical detection. Furthermore, by enhancing the activity of URG4/URGCP cyclin D1, it induces proliferation and specified target treatments can be developed [7]. In this study, URG4/URGCP cell expression, particularly in ALL, has been significantly increased. Although URG4/URGCP’s particular role in cell cycle is yet to be known, and there is statistically significant increase of URG4/URGCP gene expression in T-ALL group and in
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high-risk groups (riskier than ALL groups) and these suggest that this gene is in the proliferation inducing process. Moreover, the fact that the increase of URG4/URGCP gene expression has been found to be 204.48 times in our two cases in which remission is still not provided at the end of induction supports this observation. Due to the inadequate number of patients, there was no difference observed in URG4/URGCP gene expression for the negative patients compared with the patients with positive t (4:11) and t (9:22), involved in determining the risk groups. In conclusion, in the leukemogenesis of T-ALL during childhood, the significant expression increase of URG4/ URGCP and of the other cells in cell cycles is striking. In addition, the numeric highness of URG4/URGCP expression in the high-risk group suggests that it can be a candidate marker which may be used to determine risk groups. However, more studies with more patients are necessary to consider it a prognostic indicator and to determine its location in T-ALL leukemogenesis. In addition, they are necessary to define the disorders of gene interactions observed in cell signal transduction pathways during childhood acute leukemia, to shed light on leukemogenesis that might be specific to leukemia phenotype and to develop new target treatment models for prognosis. Acknowledgments This study is supported by Ege University Research Projects (APAK) within the scope of the project numbered 2009-TIP-34.
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