Deregulated expression of circadian clock and clock-controlledcell 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

� Springer Science+Business Media Dordrecht 2013

Abstract Circadian rhythms are endogenous and self-

sustained 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 feed-

back loops and generate circadian rhythms. The circadian

regulation extends from core clock genes to various clock-

controlled 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 simulta-

neous 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

controls in order to indicate association between deregu-

lated 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 signifi-

cantly 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

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: sqb_medgen@yahoo.com; medgen@uhs.edu.pk

W. Fatima

Department of Microbiology & Molecular Genetics, University

of the Punjab, Lahore, Pakistan

S. Mohsin

Department of Hematology, University of Health Sciences,

Lahore, Pakistan

123

Mol Biol Rep (2014) 41:95–103

DOI 10.1007/s11033-013-2841-7

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 oscilla-

tors 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, Crypto-

chrome 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 bal-

anced 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 circa-

dian 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

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 Sci-

ences, 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, Fa-

vorgen Biotech Corp., Taiwan). Native agarose gel elec-

trophoresis 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 (Gen-

Bank 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

96 Mol Biol Rep (2014) 41:95–103

123

RT-PCR as the internal control for normalization of target

gene expression. No template control (NTC) and reverse

transcriptase negative (RT-) control were always per-

formed 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 Revert-

AidTM First Strand cDNA Synthesis Kit (Cat. No. K1622,

Fermentas, Germany) in a final volume of 20 lL contain-

ing 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, reac-

tion 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).

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 per-

formed 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,

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 Ampliconsize (bp)

F-GAPDH 50-CATCTTCCAGGAGCGAGAT-30 19 327–345 52.6 ? 60.4 Exon 4–5 136

P-GAPDH 50-CTGCAAATGAGCCCCAGCCTT-30 21 441–421 57.1 - 67.0 Exon 5–6

R-GAPDH 50-GATGACCCTTTTGGCTCC-30 18 462–445 55.6 - 59.5 Exon 6

F-PER1 50-GCAGCCTCGGTTTTCTGA-30 18 3,694–3,711 55.6 ? 61.6 Exon 22 139

P-PER1 50-TGTGATGGCCTGTGTGGACTGT-30 22 3,781–3,802 54.5 ? 67.5 Exon 22–23

R-PER1 50-AGGGTGACCAGGATCTTG-30 18 3,832–3,815 55.6 - 59.9 Exon 23

F-PER2 50-CTGAAGAGGAAATGCGAGT-30 19 2,044–2,062 47.4 ? 59.1 Exon 16 140

P-PER2 50-CCACACGCTGGAGAGGCAGA-30 20 2,125–2,144 65.0 ? 67.4 Exon 16–17

R-PER2 50-GTACCTACTCCCGTGCG-30 17 2,183–2,167 64.7 - 60.2 Exon 17

F-CLK 50-CAGTCTCAAGGAAGCATTGG-30 20 1,583–1,602 50.0 ? 60.2 Exon 16 139

P-CLK 50-TCAGACCCTTCCTCAACACCAACC-30 24 1,674–1,697 54.2 ? 67.3 Exon 16–17

R-CLK 50-AGTGCTCGTATCCGTCG-30 17 1,721–1,705 58.8 - 60.0 Exon 17

F-BMAL1 50-GGAATATGTTTCTCGGCACG-30 20 1,419–1,438 50.0 ? 60.5 Exon 13 137

P-BMAL1 50-CAAAATAGCTGTTGCCCTCTGGTCT-30 25 1,488–1,464 48.0 - 66.3 Exon 13-14

R-BMAL1 50-GTCCTATGTCATCTTGGTGAA-30 21 1,555–1,535 42.9 - 59.2 Exon 14

F-CYCD1 50-CGGTGTCCTACTTCAAATGTG-30 21 331–351 47.6 ? 60.4 Exon 1 140

P-CYCD1 50-TTCCTCGCAGACCTCCAGCAT-30 21 419–399 57.1 - 67.0 Exon 1-2

R-CYCD1 50-GCGGTCCAGGTAGTTCAT-30 17 470–453 55.6 - 60.5 Exon 2

F-WEE1 50-GTGTGAAGAGGCTGGATG-30 18 2,088–2,105 55.6 ? 59.1 Exon 4 136

P-WEE1 50-CTGTTGATGAGCAGAACGCTTTGAGAG-30 27 2,151–2,177 48.1 ? 66.8 Exon 4-5

R-WEE1 50-CCTCGACGGAGTCCTC-30 16 1,243–1,258 68.8 ? 59.1 Exon 2

F-MYC 50-CCTCGACGGAGTCCTC-30 16 1,243–1,258 68.8 ? 59.1 Exon 2 140

P-MYC 50-ATCTTCTTGTTCCTCCTCAGAGTCGC-30 26 1,343–1,318 50.0 ? 66.9 Exon 2-3

R-MYC 50-CTGCCTCTTTTCCACAGAA-30 19 1,382–1,364 47.4 - 59.3 Exon 3

Mol Biol Rep (2014) 41:95–103 97

123

Human Melatonin ELISA Kit (Cat. No. CSB-E08132h,

Cusabio Biotech Co., Ltd., China) was utilized.

Statistical analysis

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.

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

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)

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

Gene Healthy individuals CLL

n 37 37

BMAL1

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

MYC

DCT (MYC–GAPDH) 5.32 ± 0.27 2.23 ± 0.19*

Relative expression 1 12.20

CyclinD1

DCT (Cyclin D1–GAPDH) 7.62 ± 0.28 3.22 ± 0.33*

Relative expression 1 26.37

WEE1

DCT (WEE1–GAPDH) 4.13 ± 0.30 6.60 ± 0.18*

Relative expression 1 -1.99

* 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 down-

regulation. Asterisk indicates significant fold change

98 Mol Biol Rep (2014) 41:95–103

123

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 sig-

nificantly 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-reg-

ulation was compared between shift-workers and non-shift-

workers, 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-shift-

workers 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 non-

shift-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 shift-

workers 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 devel-

opment. The current study was aimed to indicate associa-

tion 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 approxi-

mately 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

BMAL1

CLOCKPER1

PER2MYC

Cyclin

D1W

EE1-10

-5

0

5

10

15

20

25

30

35

40

45

50

Genes

Fol

d re

gula

tion

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 sex-

matched 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

Mol Biol Rep (2014) 41:95–103 99

123

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. Simulta-

neous 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

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-regu-

lated. C-Myc is involved in induction of G0–G1 phase

BMAL1PER1

PER2MYC

Cyclin

D1

WEE1

-20

-10

0

10

20

30

40

50

60

Shift worker

Non-Shift worker

Genes

Fol

d re

gula

tion

CLOCK

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

CLL Pat

ients

Health

y0

20

40

60

80

100

120

140

160

180

CLL PatientsHealthy

Mel

aton

in C

onc.

(pg

/ml)

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

Shift w

orker

Non-S

hift w

orker

0

20

40

60

80

100

120

140

160

180

Non-Shift worker

Shift worker

Mel

aton

in C

onc.

(pg

/ml)

Fig. 5 The bars plot graph shows a comparison of melatonin

concentrations in the serum samples of shift-workers versus non-

shift-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

100 Mol Biol Rep (2014) 41:95–103

123

transition of the cell. It is an oncogene that functions both

in the stimulation of cell proliferation and in apoptosis. C-

Myc elicits its oncogenic activity by causing immortaliza-

tion, 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 over-

expressed 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 stim-

ulates 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. Con-

sequently, 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-regula-

tion 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

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 defi-

ciency 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 mye-

loid 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 hypermethylation-

associated loss of BMAL1 also prevents the recruitment

of its natural partner, the CLOCK protein, to their com-

mon 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 pro-

moter was found to reduce breast cancer risk, and these

findings were corroborated by publicly available tissue

array data, which showed lower levels of Clock expres-

sion 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-shift-

workers. 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 con-

sidered as a biomarker of circadian disruption and has been

Mol Biol Rep (2014) 41:95–103 101

123

associated with night-shift-work and exposure to light-at-

night in both laboratory-based and field studies [42]. There

is credible evidence that a low level of melatonin is asso-

ciated 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 mani-

festation 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.

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