Mol Biol Rep (2014) 41:95–103 DOI 10.1007/s11033-013-2841-7

Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia Sobia Rana • Mustafa Munawar • Adeela Shahid Meera Malik • Hafeez Ullah • Warda Fatima • Shahida Mohsin • Saqib Mahmood

•

Received: 15 December 2012 / Accepted: 26 October 2013 / Published online: 5 November 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Circadian rhythms are endogenous and selfsustained oscillations of multiple biological processes with approximately 24-h rhythmicity. Circadian genes and their protein products constitute the molecular components of the circadian oscillator that form positive/negative feedback loops and generate circadian rhythms. The circadian regulation extends from core clock genes to various clockcontrolled genes that include various cell cycle genes. Aberrant expression of circadian clock genes, therefore, may lead to genomic instability and accelerated cellular proliferation potentially promoting carcinogenesis. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of serum melatonin levels in peripheral blood samples of 37 CLL (chronic lymphocytic leukemia) patients and equal number of age- and sex-matched healthy S. Rana  A. Shahid Department of Physiology & Cell Biology, University of Health Sciences, Lahore, Pakistan M. Munawar  H. Ullah  S. Mahmood Department of Allied Health Sciences, University of Health Sciences, Lahore, Pakistan M. Malik  S. Mahmood (&) Department of Human Genetics & Molecular Biology, University of Health Sciences, Lahore, Pakistan e-mail: [email protected]; [email protected] W. Fatima Department of Microbiology & Molecular Genetics, University of the Punjab, Lahore, Pakistan S. Mohsin Department of Hematology, University of Health Sciences, Lahore, Pakistan

controls in order to indicate association between deregulated circadian clock and manifestation of CLL. Results showed significantly down-regulated expression of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc and Cyclin D1 (P \ 0.0001) in CLL patients as compared to healthy controls. When expression of these genes was compared between shift-workers and non-shift-workers within the CLL group, the expression was found more aberrant in shift-workers as compared to non-shift-workers. However, this difference was found statistically significant for Myc and Cyclin D1 only (P \ 0.05). Serum melatonin levels were found significantly low (P \ 0.0001) in CLL subjects as compared to healthy controls whereas melatonin levels were found still lower in shift-workers as compared to non-shift-workers within CLL group (P \ 0.01). Our results suggest that aberrant expression of circadian clock genes can lead to aberrant expression of their downstream targets that are involved in cell proliferation and apoptosis and hence may result in manifestation of CLL. Moreover, shift-work and low melatonin levels may also contribute in etiology of CLL by further perturbing of circadian clock. Keywords Circadian clock  Clock genes  Clock-controlled genes  Shift-work  Melatonin  Chronic lymphocytic leukemia

Introduction Circadian rhythms are the outward manifestation of an internal timing system generated by a circadian clock that is synchronized by the day–night cycle [1]. The circadian clock proficiently coordinates the homeostatic processes of living organisms to match imposed 24-h cycles and

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influences nearly all aspects of physiology and behavior [2]. The mammalian clock system is hierarchical with a master clock controlling circadian rhythms located in the suprachiasmatic nucleus (SCN) and various slave oscillators present in peripheral organs [3]. The molecular clockwork is composed of a network of transcriptional– translational feedback loops that drive rhythmic, *24-h expression patterns of core clock components [4]. Core clock components are genes whose protein products are necessary for the generation and regulation of circadian rhythms within individual cells throughout the organism [5]. Some of core clock genes include Bmal1, Cryptochrome 1 (Cry1), Cryptochrome 2 (Cry2), Period 1 (Per1), Period 2 (Per2), Period 3 (Per3), Clock (Clk), and Casein kinase1e (CK1e) [6]. The identification of the circadian transcripts has revealed that the transcriptional circadian regulation extends beyond core clock components to include various clock-controlled genes (CCGs); genes that are under the direct or indirect transcriptional control of the clock transcription factors but are not themselves part of the clock. Regulation of CCGs is a mechanism by which the molecular clockwork controls physiological processes. The CCGs constitute about 10 % of the expressed genes in a given tissue (SCN or in peripheral tissues) [7]. CCGs may encode a variety of proteins including key regulators for cell cycle. It has been shown that expression of several cell cycle genes, including Wee1, c-Myc, CyclinD1, Gadd45, and Mdm2; oscillate in a circadian manner [8, 9]. Wee1 [8] and c-Myc [9] are directly regulated by the molecular clock via E-box elements at their promoters. E-box or Enhancer box is a DNA sequence found upstream of some promoter regions in eukaryotes and is recognized and bound by transcription factors to initiate gene transcription. Chronic lymphocytic leukemia (CLL) is characterized by proliferation and accumulation of morphologically mature but immunologically dysfunctional lymphocytes in blood, bone marrow and lymphoid tissues. No specific genetic alteration has yet been associated with this disease. In particular, CLL is not associated with reciprocal balanced chromosomal translocations, but rather with specific deletions [10] suggesting the loss of presently unidentified tumor suppressor genes. CLL cells have low proliferative rate and a prolonged life span, suggesting that their primary alteration may be a defect in apoptosis [11]. Since circadian genes regulate many biological pathways including cell proliferation and apoptosis, and CLL seems to be an apoptosis related disorder, their alteration may be directly involved in the pathophysiology of CLL. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level by quantitative (real-time) RTPCR and determination

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of serum melatonin levels by ELISA in 37 CLL patients and equal number of age- and sex-matched healthy controls in order to indicate any association between deregulated circadian clock and manifestation of CLL.

Materials and methods Samples and subjects This study was conducted after obtaining permission from Institutional Review Board of University of Health Sciences, Lahore. The study involved 37 newly diagnosed CLL patients (Binet stage A) and equal number of their age- and sex-matched healthy individuals. The patients were recruited from the oncology departments of the local tertiary care hospitals. Clinical diagnosis was based on standard morphological and immunophenotypic criteria. The informed consents were obtained from both, the patients and the healthy controls. Blood samples from CLL patients were collected before the start of their treatment regime. In order to rule out the bias of time, collection of all peripheral blood (PB) samples was carried out between 10:00 and 11:00 AM. Samples were processed within 1 h of collection for RNA and serum isolation. Quantitative RTPCR Total RNA was extracted by using FavorPrepTM Total RNA Purification Mini Kit (Cat. No. FABRK 100, Favorgen Biotech Corp., Taiwan). Native agarose gel electrophoresis was performed to assess the overall quality of total RNA. The quantity of total RNA was estimated using Nanodrop ND2000 (Thermo Scientific, USA). The first strand cDNA synthesized was used directly for amplification by polymerase chain reaction (PCR). The cDNA sequences of the four circadian clock genes namely BMAL1 (GenBank accession no. NM_001178), Clock (GenBank accession no. NM_004898), Per1 (GenBank accession no. NM_002616), Per2 (GenBank accession no. NM_022817); three cell cycle genes namely Myc (GenBank accession no. NM_002467), Cyclin D1 (GenBank accession no. NM_053056), Wee1 (GenBank accession no. NM_003390); and GAPDH (GenBank accession no. NM_002046) were determined. The specific forward and reverse primers and TaqMan probes were designed using the free online SciTools of Integrated DNA Technologies (http://eu.idtdna.com/Scitools/Applications/RealTimePCR/). The information regarding the designed primers and probes are summarized in Table 1. The designed primers and probes (labeled with appropriate fluorescent dyes) were obtained from Gene LinkTM (Hawthorne, NY, USA). Expression of GAPDH was also examined by real-time

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Table 1 Oligonucleotide primers and probes for real-time quantitative reverse transcription-polymerase chain reaction analysis of the genes under consideration Pimer

Sequence

Length

Location

GC (%)

Strand

Tm (°C)

Junction

Amplicon size (bp)

F-GAPDH P-GAPDH R-GAPDH F-PER1 P-PER1 R-PER1 F-PER2 P-PER2 R-PER2 F-CLK P-CLK R-CLK F-BMAL1 P-BMAL1 R-BMAL1 F-CYCD1 P-CYCD1 R-CYCD1 F-WEE1 P-WEE1 R-WEE1 F-MYC P-MYC R-MYC

50 -CATCTTCCAGGAGCGAGAT-30 50 -CTGCAAATGAGCCCCAGCCTT-30 50 -GATGACCCTTTTGGCTCC-30 50 -GCAGCCTCGGTTTTCTGA-30 50 -TGTGATGGCCTGTGTGGACTGT-30 50 -AGGGTGACCAGGATCTTG-30 50 -CTGAAGAGGAAATGCGAGT-30 50 -CCACACGCTGGAGAGGCAGA-30 50 -GTACCTACTCCCGTGCG-30 50 -CAGTCTCAAGGAAGCATTGG-30 50 -TCAGACCCTTCCTCAACACCAACC-30 50 -AGTGCTCGTATCCGTCG-30 50 -GGAATATGTTTCTCGGCACG-30 50 -CAAAATAGCTGTTGCCCTCTGGTCT-30 50 -GTCCTATGTCATCTTGGTGAA-30 50 -CGGTGTCCTACTTCAAATGTG-30 50 -TTCCTCGCAGACCTCCAGCAT-30 50 -GCGGTCCAGGTAGTTCAT-30 50 -GTGTGAAGAGGCTGGATG-30 50 -CTGTTGATGAGCAGAACGCTTTGAGAG-30 50 -CCTCGACGGAGTCCTC-30 50 -CCTCGACGGAGTCCTC-30 50 -ATCTTCTTGTTCCTCCTCAGAGTCGC-30 50 -CTGCCTCTTTTCCACAGAA-30

19 21 18 18 22 18 19 20 17 20 24 17 20 25 21 21 21 17 18 27 16 16 26 19

327–345 441–421 462–445 3,694–3,711 3,781–3,802 3,832–3,815 2,044–2,062 2,125–2,144 2,183–2,167 1,583–1,602 1,674–1,697 1,721–1,705 1,419–1,438 1,488–1,464 1,555–1,535 331–351 419–399 470–453 2,088–2,105 2,151–2,177 1,243–1,258 1,243–1,258 1,343–1,318 1,382–1,364

52.6 57.1 55.6 55.6 54.5 55.6 47.4 65.0 64.7 50.0 54.2 58.8 50.0 48.0 42.9 47.6 57.1 55.6 55.6 48.1 68.8 68.8 50.0 47.4

? ? ? ? ? ? ? ? ? ? ? ? ? ? -

60.4 67.0 59.5 61.6 67.5 59.9 59.1 67.4 60.2 60.2 67.3 60.0 60.5 66.3 59.2 60.4 67.0 60.5 59.1 66.8 59.1 59.1 66.9 59.3

Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon

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RT-PCR as the internal control for normalization of target gene expression. No template control (NTC) and reverse transcriptase negative (RT-) control were always performed to check reagent and DNA contamination. A positive control RTPCR was also performed every time using template RNA and GAPDH primers provided in the kit to check the fidelity of the RTPCR reaction. First strand cDNA was synthesized by using RevertAidTM First Strand cDNA Synthesis Kit (Cat. No. K1622, Fermentas, Germany) in a final volume of 20 lL containing 2 lg RNA. The tube containing RNA, Oligo (dT)18 primer and DEPC-treated water was first incubated for 5 min at 65 °C. Then rest of the components including reaction buffer, RNase inhibitor, dNTP mix and reverse transcriptase were added and incubated at 42 °C for 60 min, then the reaction was stopped by heat inactivation at 70 °C for 5 min. Prior shifting to real-time PCR, reaction was optimized through conventional RTPCR (Fig. 1). Real-time quantitative PCR was carried out in an iQTM5 Multi-color Real-Time PCR Detection System (Bio-Rad, USA) using the Maxima Probe qPCR Master Mix (Cat. No. K0232, Fermentas).

4–5 5–6 6 22 22–23 23 16 16–17 17 16 16–17 17 13 13-14 14 1 1-2 2 4 4-5 2 2 2-3 3

139

140

139

137

140

136

140

All reactions were carried out in a 25 lL final volume containing 0.3 lM each primer, 0.2 lM probe and 12.5 lL 29 Maxima Probe qPCR Master Mix. The PCR cycling parameters were set as follows: 95 °C for 10 min followed by 40 cycles of PCR reactions at 95 °C for 15 s and 60 °C for 1 min. All reactions were run in triplicates. To determine the inter-assay precision, three replicates of cDNA of each sample were run on three separate days. Intra-assay (within-run) precision was determined by calculating mean, standard deviation (SD) and coefficient of variance (CV) of the CT values for each sample and for each set of primers and probe on each day.

ELISA Enzyme-linked immunosorbent assay (ELISA) was performed to determine melatonin concentrations in the serum samples of CLL patients and their age- and sex-matched healthy controls on an automated EIA analyzer (Bio-Rad Laboratories, Hercules, CA, USA). For this purpose,

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Fig. 1 The electrophoretic gel picture shows the RTPCR product of reference and target genes resulted from an optimized RTPCR reaction. Lane 1 contain 100 bp DNA Ladder, Lane 2 contain Gapdh RTPCR product (136 bp), Lane 3 contain Bmal1 RTPCR product (137 bp), Lane 4 contains Clk RTPCR product (139 bp), Lane 5 contains Per1 RTPCR product (139 bp), Lane 6 contains Per2 RTPCR

product (140 bp), Lane 7 contains Wee1 RTPCR product (136 bp), Lane 8 contains CycD1 RTPCR product (140 bp), Lane 9 contains Myc RTPCR product (140 bp), Lane 10 contains positive control (PC) (496 bp), Lane 11 contains no template control (NTC), Lane 12 contains RT negative control (RT-ve)

Human Melatonin ELISA Kit (Cat. No. CSB-E08132h, Cusabio Biotech Co., Ltd., China) was utilized.

Table 2 The expression levels of the four circadian clock genes and three cell cycle genes as determined by real-time quantitative reverse transcription-polymerase chain reaction

Statistical analysis

Gene n

Relative gene expression levels were calculated by using Livak method [12]. Student’s t test was applied to make comparisons between the quantitative variables. P values B0.05 were regarded as being significantly different. The graphical output was generated by using GraphPad Prism version 5.00 for Windows, GraphPad Software, La Jolla, CA, USA, www.graphpad.com.

BMAL1

Results Among the 37 CLL patients, 27 (73 %) were males and 10 (27 %) were females. The age of patients ranged from 45 to 85 years with a mean age of 62.81 ± 10.84 (mean ± SD) years. Moreover, average age of male patients was 62.18 ± 11.69 and that of female patients was 64.50 ± 8.40 years. Aberrant expression of circadian clock and cell cycle genes in CLL The quantitative expression of four circadian clock and three clock-controlled cell cycle genes determined in the present study by real time quantitative RTPCR showed significantly aberrant expression of these genes in CLL patients as compared to the age- and sex-matched healthy controls (Table 2). The circadian clock genes namely hBmal1 (P \ 0.0001), hPer1 (P \ 0.0001), and hPer2 (P \ 0.0001) were found significantly down-regulated in CLL patients as compared to their healthy controls. The expression of Clock gene was found up-regulated in CLL group as compared to healthy controls but in a not

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Healthy individuals 37

CLL 37

DCT (BMAL1–GAPDH)

-1.91 ± 0.40

1.52 ± 0.28*

Relative expression

1

-3.18

CLOCK DCT (CLOCK–GAPDH)

4.44 ± 0.34

3.62 ± 0.28

Relative expression

1

2.55

PER1 DCT (PER1–GAPDH)

3.11 ± 0.29

5.96 ± 0.30*

Relative expression

1

-2.62

PER2 DCT (PER2–GAPDH)

2.38 ± 0.35

6.38 ± 0.27*

Relative expression

1

-3.27

5.32 ± 0.27 1

2.23 ± 0.19* 12.20

MYC DCT (MYC–GAPDH) Relative expression CyclinD1 DCT (Cyclin D1–GAPDH)

7.62 ± 0.28

3.22 ± 0.33*

Relative expression

1

26.37

DCT (WEE1–GAPDH)

4.13 ± 0.30

6.60 ± 0.18*

Relative expression

1

-1.99

WEE1

* P \ 0.0001 compared with normal. Results are the mean ± SE. The level of target gene was normalized to the endogenous reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to obtain the normalized circadian gene expression (DCT) value for each sample. The DCT of chronic lymphocytic leukemia cases was first related to the DCT of healthy individuals to obtain the relative threshold cycle (DDCT) and then the relative expression levels (2-DDCT) were calculated. The fold change of gene expression is calculated by taking 2-DDCT for values greater than 1 and -1/2-DDCT for values less than 1. Negative sign (-) with values of fold regulation indicates downregulation. Asterisk indicates significant fold change

Mol Biol Rep (2014) 41:95–103 50 45 40 35 30

Fold regulation

Fig. 2 The bars plot graph represents the average fold regulation values of all target genes in a population of 37 CLL patients versus 37 age- and sexmatched healthy controls. The x-axis represents the gene and the y-axis represents the fold regulation of gene expression as calculated by taking 2-DDCT for values greater than 1 and -1/2-DDCT for values less than 1

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25 20 15 10 5 0 -5

1 EE

lin yc C

W

D

1

YC M

2 PE

R

1 R PE

K C LO C

B

M

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L1

-10

Genes

statistically significant way (P [ 0.05). On the other hand, clock-controlled cell cycles genes namely hMyc (P \ 0.0001), hCyclin D1 (P \ 0.0001) were found significantly up-regulated whereas hWee1 (P \ 0.0001) was down-regulated in CLL patients as compared to healthy individuals (Fig. 2). More aberrant expression in shift workers Among a total population of 37 CLL patients, 11 (*30 %) were found shift-workers whereas all 37 healthy controls were non-shift-workers. Shift-workers included watchmen, security guards and people working in railway through nightshifts. These shift-workers had been working with nocturnal schedules with a minimum time period of 10 years to a maximum time period of 25 years. At the time of collection of blood samples, patients were newly diagnosed with CLL and most of them were either retired from the job or were on sick leave. When the expression of circadian clock and cell cycle genes in terms of fold-regulation was compared between shift-workers and non-shiftworkers, a more severely aberrant expression was found in shift-workers as compared to non-shift-workers within the CLL group. Bmal1, Per1 and Per2 showed greater fold down-regulation in shift-workers as compared to non-shiftworkers within the CLL group. Similarly, Myc and Cyclin D1 showed greater fold up-regulation in shift-workers as compared to non-shift-workers (Fig. 3). However, when t test was applied, only Myc (P \ 0.05) and Cyclin D1 (P \ 0.05) significantly showed greater fold up-regulation whereas Bmal1, Per1 and Per2 were not significantly altered (P [ 0.05) in shift-workers as compared to nonshift-workers within the CLL group.

Low melatonin concentrations in CLL subjects and shift workers The concentrations of melatonin (pg/mL) in the serum samples of CLL patients were found significantly (P \ 0.05) lower (101.57 ± 3.65, mean ± SEM) as compared to their age- and sex-matched healthy controls (141.14 ± 7.77, mean ± SEM) as shown in Fig. 4. Furthermore, serum melatonin concentrations were also found significantly (P \ 0.05) lower (86.89 ± 5.60, mean ± SEM) in shiftworkers as compared to non-shift-workers (107.78 ± 4.11, mean ± SEM) within the CLL study group (Fig. 5).

Discussion Since many genes involved in cell cycle are under the control of circadian clock, maintaining the circadian rhythms can be a critical control point for cancer development. The current study was aimed to indicate association between deregulated circadian clock and manifestation of CLL. CLL is considered to be mainly a disease of the elderly, with a median age at diagnosis of 70 years [13] and it has been reported that the CLL disorder is more common in men with a male to female ratio of approximately 2:1 [14]. The same observations remained almost true in the present study as CLL patients included in the current study mostly constituted elderly population with the age ranged from 45 to 85 years (mean age of 62.81 ± 10.84) having a male dominance with male to female ratio of 2.7:1. According to the most well characterized positive feedback loop, the transcription of Per1 and Per2 genes is

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Mol Biol Rep (2014) 41:95–103 60 Shift worker

50

Non-Shift worker

40

Fold regulation

Fig. 3 The bars plot graph shows a comparison of the average fold regulation values of study genes in shift-workers versus non-shift-workers within CLL population. The x-axis represents the gene and the y-axis represents the fold regulation of gene expression as calculated by taking 2-DDCT for values greater than 1 and -1/2-DDCT for values less than 1

30

20

10

0

1 W

EE

D lin yc

M

PE

1

YC

2 R

1 R PE

C LO

C

C

B

M

A

-20

K

L1

-10

Genes 180 CLL Patients Healthy

160 140

Melatonin Conc. (pg/ml)

120 100 80 60 40 20

Shift worker

160

Non-Shift worker

140 120 100 80 60 40 20

r ke or w hi

ft

ift

H

w

ea

or

lth

ke

y

N

on

-S

s nt tie C

LL

Pa

r

0

0

Sh

Melatonin Conc. (pg/ml)

180

Fig. 4 The bars plot graph shows a comparison of melatonin concentrations in the serum samples of CLL patients versus their age- and sex-matched healthy controls. Melatonin concentrations in CLL patients were found significantly (P B 0.05) lower as compared to healthy controls

directly activated by BMAL1/CLOCK heterodimers [15]. That is why, in our study, down-regulation of Bmal1 is accompanied with down-regulation of Per1 and Per2 in CLL patients as compared to healthy controls. Simultaneous down-regulation of Bmal1 and Period genes has also been reported in chronic myeloid leukemia [16] and head and neck squamous cell carcinoma [17]. Disturbances in the periodic expression of Cry1, Per1 and Per2 genes have also been reported recently [18]. A number of studies revealed that the loss and deregulation of Per genes is common in cancer. Diminished expression levels of Per1

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Fig. 5 The bars plot graph shows a comparison of melatonin concentrations in the serum samples of shift-workers versus nonshift-workers within the CLL group. Melatonin concentrations in shift-workers were found significantly (P B 0.05) lower as compared to non-shift-workers within the CLL group

and Per2 mRNA have been reported in human colorectal cancer [19]. Mutation of these 2 core clock genes has also been identified in breast and colorectal cancers [20]. On the other hand, over-expression of Per1 or Per2 inhibits cancer cell growth in culture as well as in animals [21–23]. All this information indicates tumor suppressive nature of Per1 and Per2 [24]. Myc, Cyclin D1 and Wee1 are clock-controlled cell cycle genes. In the current study, Myc and Cyclin D1 are found up-regulated whereas Wee1 is found down-regulated. C-Myc is involved in induction of G0–G1 phase

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transition of the cell. It is an oncogene that functions both in the stimulation of cell proliferation and in apoptosis. CMyc elicits its oncogenic activity by causing immortalization, and to a lesser extent the transformation of cells, in addition to several other mechanisms [25]. Virtually, all types of human cancer manifest high frequencies of amplification of the c-Myc gene or over-expression of its protein product [26]. Cyclin D1 is another oncogene that drives cell cycle progression (G1-S phase transition); it acts as a growth factor sensor to integrate extracellular signals with the cell cycle machinery, though it may also promote apoptosis [25]. Deregulation of Cyclin D1 gene expression and increased proliferation are hallmarks of a number of proliferative diseases, including cancer. Cyclin D1 is overexpressed in many types of human cancer, with gene amplification in some cases [27]. WEE1 is a cell cycle kinase that controls the timing of G2–M transition. It is activated by ongoing DNA replication or by the presence of DNA damage and inactivates Cdc2/cyclin B through phosphorylation resulting in the delay of mitosis or arrest of the cell cycle at the G2–M interface [28]. In case of Wee1 inhibition, cells would not undergo cell cycle arrest and mitosis would continue. Reduced expression of Wee1 has been reported in a number of cancers including colon carcinoma [29] and non-small-cell lung cancer (NSCLC) [30]. Wee1 and c-Myc are directly regulated by the BMAL1:CLOCK heterodimer via the E-box elements at their promoters [8, 9]. Normally, the binding of BMAL1:CLOCK to the E-boxes of c-Myc promoter inhibits the transcription of this gene and the binding of BMAL1:CLOCK to the E-boxes of Wee1 promoter stimulates the transcription of this gene. Thus, reduced level of BMAL1 may lead to up-regulation of c-Myc transcription and down-regulation of Wee1 transcription that seems to be the case in the current study. It has been indicated that Cyclin D1 is under circadian control in vivo [9]. Cyclin D1 can be directly reduced [31] or indirectly induced by c-Myc [32]. Cyclin D1 expression was reported to be arrhythmic and significantly elevated at most times in the bones of Per1-/-; Per2m/m mice than in wild-type (wt) mice. BMAL1/CLOCK was found to inhibit the promoter activity of c-Myc, a critical regulator of Cyclin D1. Consequently, c-Myc expression was elevated in Per1-/-; Per2m/m bones at most time points studied and in Per1-/-; Per2m/m osteoblasts. Thus, one mechanism whereby clock genes inhibit osteoblast proliferation is the down-regulation of c-Myc expression, although other mechanisms may exist [33]. In a recent study, down regulation of Bmal1 gene expression was found to accelerate cell proliferation in vitro and promote tumor growth in mice. Suppressing Bmal1 expression in murine colon cancer cells (C26) and fibroblast cells (L929) was reported to cause decreased apoptosis. Loss of Bmal1 led to the reduced expression of

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Per1, Per2, Per3, Wee1 and p53. The expression of p21 and c-Myc was also found up-regulated in certain cell lines (IEC—intestinal epithelial cells). However, BMAL1 deficiency was reported to increase the protein levels of Cdc2, Cyclin B1, Cyclin D1 and Cyclin E [34]. Bmal1 epigenetic inactivation contributes to the development of hematologic malignancies such as diffuse large B-cell lymphoma and acute lymphocytic and myeloid leukemias by disrupting the cellular circadian clock. Bmal1 epigenetic inactivation impairs the characteristic circadian clock expression pattern of genes such as c-Myc with a loss of BMAL1 occupancy in their respective promoters. Furthermore, the DNA hypermethylationassociated loss of BMAL1 also prevents the recruitment of its natural partner, the CLOCK protein, to their common targets [35]. However, in the current study it is not known whether the down-regulation of Bmal1 in CLL was the result of any epigenetic phenomena. Another study showed that hypermethylation in the Clock promoter was found to reduce breast cancer risk, and these findings were corroborated by publicly available tissue array data, which showed lower levels of Clock expression in healthy controls relative to normal or tumor tissue from breast cancer patients [36]. In our study, Clock expression was also found higher in CLL patients as compared to healthy controls but it was statistically insignificant (P [ 0.05). Further investigations in a bigger study population may help to get the more lucid picture about the expression of Clock gene in CLL. In current study, among 37 CLL patients, *30 % were shift-workers. Shift-work has been reported as a risk factor for cancer in many studies [37]. Women working more than 20 years of rotating nightshifts were found to have a significantly increased risk of endometrial cancer [38]. A significant association between rotating shift-work and prostate cancer incidence among Japanese male workers has also been reported [39]. According to another study full time rotating shift-work was found to be associated with increased risk of prostate cancer [40]. Non-Hodgkin’s lymphoma was found to be modestly associated with nighttime work among men with high exposure [41]. In the current study, expression levels of the genes were found more aberrant in shift-workers as compared to non-shiftworkers. Bmal1, Per1 and Per2 were more down-regulated whereas Myc and Cyclin D1 were more up-regulated in shift-workers as compared to non-shift-workers within the CLL group. When serum melatonin concentrations were compared between CLL patients and healthy controls, significantly low melatonin levels were found in CLL patients. Moreover, melatonin levels were found still lower in shift-workers as compared to non-shift-workers within the CLL group. Circulating melatonin level can be considered as a biomarker of circadian disruption and has been

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associated with night-shift-work and exposure to light-atnight in both laboratory-based and field studies [42]. There is credible evidence that a low level of melatonin is associated with an increased risk of prostate [43] and breast cancer [44]. Conclusion Our results indicate that deregulated expression of core clock genes may result in deregulated expression of CCGs that are involved in cell proliferation and apoptosis and hence may play a role in etiology of CLL. Furthermore, shift-work and low melatonin levels may also contribute in further perturbing of circadian clock and hence in manifestation of CLL. Additional research needs to be carried out to elucidate the mechanisms by which shift-work and low melatonin levels contribute in perturbing the circadian clock in CLL. Acknowledgments This work was supported by an HEC (Higher Education Commission of Pakistan)-funded Project ‘‘Centre for Research in Endocrinology and Reproductive Sciences’’ (CRERS) in University of Health Sciences, Lahore, Pakistan.

References 1. Reppert SM, Weaver DR (2002) Coordination of circadian timing in mammals. Nature 418:935–941 2. Hastings MH, Reddy AB, Maywood ES (2003) A clockwork web: circadian timing in brain and periphery, in health and disease. Nat Rev Neurosci 4:649–661 3. Geyfman M, Andersen B (2009) How the skin can tell time. J Invest Dermatol 129:1063–1066 4. Shearman LP, Sriram S, Weaver DR, Maywood ES, Chaves I, Zheng B, Kume K, Lee CC, van der Horst GT, Hastings MH, Reppert SM (2000) Interacting molecular loops in the mammalian circadian clock. Science 288:1013–1019 5. Takahashi JS (2004) Finding new clock components: past and future. J Biol Rhythms 19:339–347 6. Fu L, Lee CC (2003) The circadian clock: pacemaker and tumor suppressor. Nat Rev Cancer 3:350–361 7. Duffield GE (2003) DNA microarray analyses of circadian timing: the genomic basis of biological time. J Neuroendocrinol 15:991–1002 8. Matsuo T, Yamaguchi S, Mitsui S, Emi A, Shimoda F, Okamura H (2003) Control mechanism of the circadian clock for timing of cell division in vivo. Science 302:255–259 9. Fu L, Pelicano H, Liu J, Huang P, Lee C (2002) The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111:41–50 10. Do¨hner H, Stilgenbauer K, Do¨hner M (1999) Chromosome aberrations in B-cell chronic lymphocytic leukemia: reassessment based on molecular cytogenetic analysis. J Mol Med 77:266–281 11. Caligaris-Cappio F, Hamblin TJ (1999) B-cell chronic lymphocytic leukemia: a bird of a different feather. J Clin Oncol 17:399–408 12. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408

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Mol Biol Rep (2014) 41:95–103 13. Smith A, Howell D, Patmore R (2011) Incidence of haematological malignancy by sub-type: a report from the Haematological Malignancy Research Network. Br J Cancer 105:1684–1692 14. Sgambati M, Linet MS, Devesa SS (2001) Chronic lymphocytic leukemia epidemiological, familial, and genetic aspects. In: Cheson BD (ed) Chronic lymphoid leukemia s basic and clinical oncology. Marcel Dekker, New York, pp 33–62 15. Takahashi JS, Hong HK, Ko CH, McDearmon EL (2008) The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat Rev Genet 9:764–775 16. Yang MY, Chang JG, Lin PM, Tang KP, Chen YH, Lin HY, Liu TC, Hsiao HH, Liu YC, Lin SF (2006) Down-regulation of circadian clock genes in chronic myeloid leukemia: alternative methylation pattern of hPER3. Cancer Sci 97:1298–1307 17. Hsu CM, Lin SF, Lu CT, Lin PM, Yang MY (2012) Altered expression of circadian clock genes in head and neck squamous cell carcinoma. Tumour Biol 33:149–155 18. Eisele L, Prinz R, Klein-Hitpass L, Nu¨ckel H, Lowinski K, Thomale J, Moeller LC, Du¨hrsen U, Du¨rig J (2009) Combined PER2 and CRY1 expression predicts outcome in chronic lymphocytic leukemia. Eur J Haematol 83:320–327 19. Mostafaie N, Ka´llay E, Sauerzapf E, Bonner E, Kriwanek S, Cross HS, Huber KR, Krugluger W (2009) Correlated downregulation of estrogen receptor beta and the circadian clock gene Per1 in human colorectal cancer. Mol Carcinog 48:642–647 20. Sjo¨blom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, Mandelker D, Leary RJ, Ptak J, Silliman N, Szabo S, Buckhaults P, Farrell C, Meeh P, Markowitz SD, Willis J, Dawson D, Willson JK, Gazdar AF, Hartigan J, Wu L, Liu C, Parmigiani G, Park BH, Bachman KE, Papadopoulos N, Vogelstein B, Kinzler KW, Velculescu VE (2006) The consensus coding sequences of human breast and colorectal cancers. Science 314:268–274 21. Gery S, Komatsu N, Baldjyan L, Yu A, Koo D, Koeffler HP (2006) The circadian gene per1 plays an important role in cell growth and DNA damage control in human cancer cells. Mol Cell 22:375–382 22. Hua H, Wang Y, Wan C, Liu Y, Zhu B, Yang C, Wang X, Wang Z, Cornelissen Guillaume G, Halberg F (2006) Circadian gene mPer2 overexpression induces cancer cell apoptosis. Cancer Sci 97:589–596 23. Gery S, Gombart AF, Yi WS, Koeffler C, Hofmann WK, Koeffler HP (2005) Transcription profiling of C/EBP targets identifies Per2 as a gene implicated in myeloid leukemia. Blood 106:2827–2836 24. Yang X, Wood PA, Ansell C, Hrushesky WJ (2009) Circadian time-dependent tumor suppressor function of period genes. Integr Cancer Ther 8:309–316 25. Liao DJ, Thakur A, Wu J, Biliran H, Sarkar FH (2007) Perspectives on c-Myc, Cyclin D1, and their interaction in cancer formation, progression, and response to chemotherapy. Crit Rev Oncog 13:93–158 26. Dang CV, O’Donnell KA, Zeller KI, Nguyen T, Osthus RC, Li F (2006) The c-Myc target gene network. Semin Cancer Biol 16:253–264 27. Fu M, Wang C, Li Z, Sakamaki T, Pestell RG (2004) Minireview: Cyclind1: normal and abnormal functions. Endocrinology 145:5439–5447 28. Sancar A, Lindsey-Boltz LA, Unsal-Kac¸maz K, Linn S (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73:39–85 29. Backert S, Gelos M, Kobalz U, Hanski ML, Bo¨hm C, Mann B, Lo¨vin N, Gratchev A, Mansmann U, Moyer MP, Riecken EO, Hanski C (1999) Differential gene expression in colon carcinoma cells and tissues detected with a cDNA array. Int J Cancer 82:868–874

Mol Biol Rep (2014) 41:95–103 30. Yoshida T, Tanaka S, Mogi A, Shitara Y, Kuwano H (2004) The clinical significance of Cyclin B1 and Wee1 expression in nonsmall-cell lung cancer. Ann Oncol 15:252–256 31. Mateyak MK, Obaya AJ, Sedivy JM (1999) C-Myc regulates cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle progression at multiple independent points. Mol Cell Biol 19:4672–4683 32. Marhin WW, Hei YJ, Chen S, Jiang Z, Gallie BL, Phillips RA, Penn LZ (1996) Loss of Rb and Myc activation co-operate to suppress cyclin D1 and contribute to transformation. Oncogene 12:43–52 33. Fu L, Patel MS, Bradley A, Wagner EF, Karsenty G (2005) The molecular clock mediates leptin-regulated bone formation. Cell 122:803–815 34. Zeng ZL, Wu MW, Sun J, Sun YL, Cai YC, Huang YJ, Xian LJ (2010) Effects of the biological clock gene Bmal1 on tumour growth and anti-cancer drug activity. J Biochem 148:319–326 35. Taniguchi H, Ferna´ndez AF, Setie´n F, Ropero S, Ballestar E, Villanueva A, Yamamoto H, Imai K, Shinomura Y, Esteller M (2009) Epigenetic inactivation of the circadian clock gene BMAL1 in hematologic malignancies. Cancer Res 69:8447–8454 36. Hoffman AE, Yi CH, Zheng T, Stevens RG, Leaderer D, Zhang Y, Holford TR, Hansen J, Paulson J, Zhu Y (2010) CLOCK in breast tumorigenesis: evidence from genetic, epigenetic, and transcriptional profiling analyses. Cancer Res 70:1459–1468 37. Straif K, Baan R, Grosse Y, Secretan B, El Ghissassi F, Bouvard V, Altieri A, Benbrahim-Tallaa L, Cogliano V (2007) Carcinogenicity of shift-work, painting, and fire-fighting. Lancet Oncol 8:1065–1066

103 38. Viswanathan AN, Hankinson SE, Schernhammer ES (2007) Night shift work and the risk of endometrial cancer. Cancer Res 67:10618–10622 39. Kubo T, Ozasa K, Mikami K, Wakai K, Fujino Y, Watanabe Y, Miki T, Nakao M, Hayashi K, Suzuki K, Mori M, Washio M, Sakauchi F, Ito Y, Yoshimura T, Tamakoshi A (2006) Prospective cohort study of the risk of prostate cancer among rotatingshift workers: findings from the Japan collaborative cohort study. Am J Epidemiol 164:549–555 40. Conlon M, Lightfoot N, Kreiger N (2007) Rotating shift work and risk of prostate cancer. Epidemiology 18:182–183 41. Lahti TA, Partonen T, Kyyro¨nen P, Kauppinen T, Pukkala E (2008) Night-time work predisposes to non-Hodgkin lymphoma. Int J Cancer 123:2148–2151 42. Mirick DK, Davis S (2008) Melatonin as a biomarker of circadian dysregulation. Cancer Epidemiol Biomarkers Prev 17:3306–3313 43. Bartsch C, Bartsch H, Schmidt A, Ilg S, Bichler KH, Flu¨chter SH (1992) Melatonin and 6-sulfatoxymelatonin circadian rhythms in serum and urine of primary prostate cancer patients: evidence for reduced pineal activity and relevance of urinary determinations. Clin Chim Acta 209:153–167 44. Schernhammer ES, Hankinson SE (2009) Urinary melatonin levels and postmenopausal breast cancer risk in the Nurses’ Health Study cohort. Cancer Epidemiol Biomarkers Prev 18:74–79

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