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Int J Biol Med Res. 2014; 5(3): 4150-4155
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BIOLOGICAL AND MEDICAL RESEARCH
Original Article
Sex Chromatin Frequency Variation among Breast Cancer Patients and Normal
Females of Two Reproductive Stages from Bengalee Hindu Females
Koel Mukherjeea, Pulamaghatta N. Venugopala, Malay Kumar Barmanb, Arup Ratan Bandyopadhyayc
a
Anthropological Survey of India, North West Regional Centre, Dehradun
North Bengal Medical College, Sushruta Nagar, Dist-Darjeeling, West Bengal
c
Department of Anthropology, University College of Science, Technology & Agriculture, University of Calcutta, Kolkata
b
ARTICLE INFO
ABSTRACT
Keywords:
Sex Chromatin
XIST
BRCA1
Bengalee Hindu Females
X chromosome inactivation refers to the developmentally regulated process of silencing gene
expression from all but one X chromosome per cell in female mammals in order to equalize the
levels of X chromosome derived gene expression between the sexes. In females, X chromosome
inactivation (XCI) begins with the expression of the XIST gene from the X chromosome
destined to be inactivated (Xi) and the coating of XIST RNA in cis. The apparent cytological
overlap between BRCA1 and XIST RNA across the Xi raised the possibility of a direct role of
BRCA1 in localizing XIST. The present study was conducted to evaluate the comparison of
prevalence of sex chromatin in Bengalee Hindu Breast Cancer patients with Normal Bengalee
Hindu Caste females, belong to two different reproductive stages viz. Menstruating females
and Menopausal females. Materials for the present study consisted of the samples of buccal
smears of 75 normal females (40 individuals from menstruating stage and 35 individuals from
Menopausal stage) and 68 females of carcinoma of breast belonging to stage II, III, and IV. One
hundred cells from each individual will be studied to ascertain the modal rate of incidences of
sex chromatin. Our results revealed a significant difference between mean prevalence of Sex
Chromatin among Breast Cancer patients, Menstruating normal and Menopausal normal
females. Further, One-way ANOVA revealed a significant difference between the mean on Sex
Chromatin frequencies among different stages of Breast cancer patients. This result is
indicating reactivation of inactive X chromosome in case of malignancy. This result suggested
the prognostic value of prevalence of sex chromatin in Breast Cancer patients.
c Copyright 2010 BioMedSciDirect Publications IJBMR -ISSN: 0976:6685. All rights reserved.
1. Introduction
In all female mammals one or other of the two X chromosomes
is inactive in all somatic cells [1,2]. The presence of an inactive X (Xi)
chromosome in female cells was first observed at the cytological
level by Barr and Bertram in 1949 as a 1µ heteropycnotic body mass
that was often found at the nuclear periphery or within the
perinuclear region[3,4]. In mammalian females; dosage
compensation of X-linked genes is achieved by random inactivation
of one of the two X chromosomes in somatic cells. The major genetic
locus proposed to control the X chromosome inactivation process is
the X inactivation center (XIC). XIC is defined as a region of the X
chromosome from which a currently ill-defined inactivation signal
exerts its effect in cis along the chromosome; derivative X
chromosomes lacking this XIC are unable to become inactivated [5].
* Corresponding Author : Koel Mukherjee
Anthropological Survey of India
North West Regional Centre,Dehradun-248195
9830515315
koelanthro@gmail.com
c Copyright
2010 BioMedSciDirect Publications. All rights reserved.
In search of X inactivation, the mechanism by which X-linked
gene expression is equalized between XX females and XY males
revealed the antisense gene Tsix determines X chromosome choice
and represses the noncodig silencer, Xist [6,7,8]. This process of X
chromosome inactivation (XCI) is a remarkable example of long
ra n g e , m o n o - a l l e l i c g e n e s i l e n c i n g a n d fa c u l t a t ive
heterochromatin formation [9,10,11,12] even in female human
embryonic stem cells [13]. Xist provides to functionally define
epigenetic transitions in development, to understand cell identity,
pluripotency and stem cell differentiation [14].
The study of X inactivation may also provide insight into cancer
biology, as two active Xs have been found in many human breast
and ovarian tumors [15]. Additionally, the X-inactivation process
can extend beyond the scope of X-linked genes and be applied to
many human disorders involving imprinted genes—genes
expressed from only one of two parental chromosomes that are
Koel Mukherjee et.al Int J Biol Med Res. 2014; 5(3): 4150-4155
4151
apparently also regulated by noncoding RNAs [16]. Without a
doubt, X inactivation represents a great model system with which to
study a broad range of developmental and epigenetic
processes—those involving stable gene expression without
changes to the underlying DNA sequence.
It is initially believed that the X chromosome inactivation is
stably maintained in all progeny cells. However, various studies
showing alteration of X chromatin frequency during age changes,
different phases of menstrual cycle, pregnancy [17] and neoplasia
[18] suggested skewed X inactivation (non-random) and
reactivation of inactive X chromosome. Skewing of X inactivation
has strong implication in biological consequences for female
individual. Because XCI is stable once established, clonal expansion
of somatic cells as occur in the cancer- result in a cell population
with extremely skewed X inactivation [19]. This is often used to
assess tumor clonality [20]. It is known that the two X chromosomes
are active in oocytes [21,22,23], indicating that the inactive X
chromosome must be reactivated during germ cell development
[24]. Some recent findings indicate that reactivation of the inactive
X chromosome occurs at least twice during mammalian
development, once in the epiblast cell lineage at the periimplantation stage and once in the Primordial Germ Cells at the
midgestation stage, and that the reactivation of the inactive X
chromosome appears to be tightly correlated with major genomic
reprogramming events occurring during mammalian development
[25,26]. The reactivation of inactive X chromosome was also
observed whenever the body was under physiological stress
especially in neoplasia [18]. Because female mammalian cells only
have one active X chromosome, either loss of heterozygosity at the
active X chromosome or skewed X chromosome inactivation may
result in the loss of the function of an X-linked tumor suppressor
gene and may lead to the cancer predisposition [27].
In the view of the above, the present study is an attempt to
compare the prevalence of sex chromatin changes, if any, in breast
cancer patients with normal healthy females of two reproductive
stages.
Materials and Methods
A total of 143 females were included in the study, out of which
75 of normal females and 68 were diagnosed as carcinoma breast
cancer patients belongs to Bengalee Hindu community. Further,
normal females was categorised in to Menstruating females and
Menopausal females and carcinoma breast cancer patients
categorised in to different stages viz. Stage II, III and IV. The
diagnosis of these breast cancer patients was confirmed by
histopathologic biopsy and for both the group one hundred (100)
cells from each individual was studied to ascertain the modal rate of
incidences of sex chromatin.
A structured schedule was used to collect the data on the
demography, life style pattern, reproductive history and clinical
details from breast cancer patients.
Buccal smear samples have been collected with the help of
foam-tip buccal cell collection swab. The scrapped material was
spread quickly over the glass slide. Fixation and staining
(CarbolFuchsin) of slides have done by using standard technique
[28]. For each individual 100 cells will be considered at random.
Slides were scanned through 10×40 resolution. Appropriate
statistical tests were carried on using in SPSS software (version
16.0).
Results
A total of 143 samples were analysed for the present study.
Frequency of sex chromatin distribution indicated that a majority of
participants (44%) were showing presence of Sex chromatin in 2130 cells, 21% of them showing in 41-50 cells, 18% of them showing
in 11-20 cells, 13% of them showing in 31-40 cell and only 5% of
subjects showing in >50 cell. Group (Breast cancer and Normal
females) wise distribution indicated that in breast cancer patients,
the maximum patients were showing the Sex chromatin in 21-30
cells followed by 11-20 cells and only 4% of them showing in 31-40
cells. In case of Normal females, the maximum participants showing
the presence of Sex chromatin in 41-50 cells followed by 21-30
cells, 31-40 cells, and >50 cells. In case of breast cancer none of them
were showing the presence of Sex chromatin in more than 40 cells
and in case of normal females none of them were showing presence
of Sex chromatin in less than 20 cells. Further, contingency table
analysis revealed a significant association between frequency of sex
chromatin distribution and Groups (breast cancer patients and
normal females) (CC=0.573; P=0.000). In other words the presence
of Sex chromatin in breast cancer patients is comparatively lower
than the normal females (Table 1).
The frequency distribution of sex chromatin with menstruating
and menopausal females also found to be significant (CC=0.672;
P=0.000) and it is found that in menstruating females the frequency
of Sex chromatin were more and in Menopausal females the
frequency was less, none of the menopausal females were shown
the presence of Sex chromatin in more than 40 cells. However, the
non significant result was observed in case of frequency
distribution of sex chromatin with different stages of breast cancer.
The non significant was found due to the higher ranges
classification of the frequency distribution of sex chromatin, since,
minimum and maximum Sex chromatin found in breast cancer
patient were 11 and 33 respectively. As a result, later for the micro
level observation of frequency distribution of sex chromatin in
breast cancer stages the range classification has been reduced and
reanalysed (table.2). Further, it is found to be significant difference
in frequency distribution of sex chromatin with different stages of
breast cancer (CC=0.500; P=0.004). It is clear from the table, as the
stages of the breast cancer increases the frequency of sex chromatin
decreases.
Koel Mukherjee et.al Int J Biol Med Res. 2014; 5(3): 4150-4155
4152
The average frequency sex chromatin of breast cancer subject and normal subject were found to be 22.57 and 37.39 respectively. Student
“t” test revealed a significant difference between these average frequency sex chromatin with t value of 12.251 at significance of 0.000 level. In
other words the average frequency of sex chromatin in normal females was significantly increased in 14.82 cells than breast cancer patients.
Further, “t” test revealed a significant difference in the frequency of sex chromatin between menstruating and menopausal females (t=16.458;
P=0.000). The average frequency of sex chromatin significantly decreased in menopausal with 15.73 cells (Table 3).
Koel Mukherjee et.al Int J Biol Med Res. 2014; 5(3): 4150-4155
4153
One-way ANOVA revealed a significant difference between
average frequency sex chromatin among breast cancer,
menstruating and menopausal females of Bengalee Hindu
population. The average frequency of sex chromatin varies from
22.44 to 44.73 among different groups of female participants and
which is found to be significant (F=307.015; p=0.000). The average
frequency chromatin of breast cancer, menstruating and
menopausal females were 22.44, 44.73 and 29.00 cells respectively.
Further, Scheffe's post hoc test revealed that each mean difference
between different groups of female subjects was significantly
different. Further, average frequency sex chromatin frequency
varies from 20.13 to 25.60 among different stages of breast cancer,
which is found to be highly significant. One-way ANOVA revealed a
significant difference between these average sex chromatin
frequencies with F value of 9.829 and significance level of 0.000
level. Further, Scheffe's post hoc test mentioned at superscript in
alphabets which revealed that different alphabets are statistically
significant in other words mean difference between tribes was
significantly different and same alphabets are found to be
significantly no different (Table 3).
Discussion
Although higher prevalence of sex chromatin has been reported
in breast cancer patients [29], again a study indicated no significant
change in X chromatin frequency in case of breast cancer patients
[30]. Apart from these, earlier studies have shown significantly
lower prevalence of sex chromatin in different malignancy
[1,31,32,33,34,35,36]. The similar results indicating lower
prevalence of sex chromatin have been revealed in the studies on
carcinoma for example, esophageal cancer [37], breast cancer
[38,39].
In this context, the present study also revealed comparatively
lower prevalence of sex chromatin in breast cancer patients than
normal females which corroborates the earlier studies [38,40,39].
In our present study the prevalence of sex chromatin in breast
cancer patients have been shown significantly lower value in
menstruating as well as menopausal patients as compared to the
normal menstruating and menopausal females. The reason behind
the comparatively lower prevalence of sex chromatin in breast
cancer patients may be the reactivation of inactive X chromosome.
In eutherian mammals, the inactive X chromosome (Xi) differs
from its active homologue (Xa) in a number of ways, including
increased methylation of selected CpGs, replication late in S-phase,
expression of the Xist gene with binding of Xist RNA and
underacetylation of core histones [41]. DNA methylation and
histone acetylation plays an important role in the stability and
maintenance of gene silencing in inactive X chromosome [42,43]. A
strong correlation between DNA hypermethylation, transcriptional
silence and tightly compacted chromatin has been established in
many different research works [44,45,46]. Similarly, many research
works had shown that inactive X in female individuals contains
underacetelated H4 because histone under acetylation plays an
important role in the stabilization of inactive state of a gene [47,41].
But alteration in DNA methylation and Acetylation are common
in various types of tumors as well as development [47].
Hypomethylation of DNAs causes transcriptional activation and has
been hypothesized to contribute to oncogenesis by activation of
oncogenes, found in various kinds of cancers such as breast cancer,
cervical cancer, brain cancer [48,49]. The DNA methylation is
correlated with deacetylation of histone H3 and H4, along with
shifts in histone methylation pattern [50,51]. In this way, the
pattern of histone modification modulate a landscape that
organizes and maintains the integrity of the nuclear architecture
while establishing and enabling the expression pattern of specific
genes within chromatin regions [52]. In the work done by Roh et al.
in the year of 2005 and 2007, high resolution genome wide mapping
has revealed high levels of histone H3 acetylation in carcinogenic
cells.[53,54]
Recently, research studies have demonstrated quantitative
variation in prevalence of sex chromatin can be used as a prognostic
tool in case of breast cancer. Ghosh et al. have shown a positive
significant correlation between sex chromatin incidence and a five
year survival time or disease free interval of distant metastasis.[55]
Longitudinal studies on cancer patients, however, demonstrated
initial higher prevalence of sex chromatin and gradual lower
frequency of sex chromatin at the final stage and in addition to that,
the study by Perry in 2005 on evaluation of breast tumor sex
chromatin (barr body) as an index of survival and response to
pituitary ablation reported that the high tumor sex chromatin
counts were associated with long survival after treatment, and low
counts with short survival[40]. Another work done by Wacker &
Charles in 2006 revealed a higher frequency of sex chromatin in
patients who survived longed than 8 years than who expired earlier
[4]. The quantitative features of chromatin structure in the
prognosis of breast cancer and the results demonstrated that the
criteria used enable prediction of prognosis with higher accuracy
(92%) [56]. The significance of sex chromatin test consists in the
fact that one may judge the rate of tumor growth by the sex
chromatin content in a small volume of biopsy material. Therefore,
the sex chromatin test is an index of the growth rate (proliferative
activity) of the examined tumor. By this test it is possible to
determine the degree of the tumor progression, to assess the
mitotic activity in the small pieces of the biopsy material. The sex
chromatin test may be an additional method for differential
diagnosis of malignant tumors [57].
Conclusion
In our present study, it has been revealed that sex chromatin
prevalence in breast cancer patients is comparatively lower than
apparently normal healthy female individual. Furthermore the
frequency of sex chromatin varies among the stages of breast cancer
patients also. Then the findings of the present study signifies that
the alteration in the pattern of DNA methylation and Histon
acetylation in cancer cells may be the cause behind lower
prevalence of sex chromatin in breast cancer females than normal
females. Furthermore, the present study is being the first attempt
from Bengalee Hindu Caste females that whether the prevalence of
sex chromatin has any prognostic value for Bengalee Hindu Caste
breast cancer patients or not.
Koel Mukherjee et.al Int J Biol Med Res. 2014; 5(3): 4150-4155
4154
Acknowledgements:
The authors are grateful to Dr. Gautam Das, Associate Professor
of Calcutta National Medical College and Dr Ranen kumar Aich,
Associate Professor of Nilratan Sarkar Medical College and Hospital
for their cooperation in connection with the collection of buccal
smear samples of Breast cancer patients. The authors express
heartfelt gratitude to all the participants for their sincere
cooperation and generous help in collecting the data for this study.
Concerning the preparation of slides for microscopy, the authors
remain thankful to Dr Indrajit Das Gupta and Jayita RayTapadar,
Department of Anthropology, University of Calcutta.
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c Copyright 2010 BioMedSciDirect Publications IJBMR -ISSN: 0976:6685.
All rights reserved.