Role of chromosomal fragile site in the identification and characterization of human malignancy

About Author: Rinki Verma (Research fellow)
Center of Experimental Medicine and Surgery,
Institute of Medical science
Banaras Hindu University
Varanasi - 221005

Abstract:
In 1965 , firstly fragility was reported in cells of a femeal previously irradiated and described as non-random human chromosome called “ Fragile sites” are heritable specific chromosome loci that exhibit an increased frequency of gaps, poor staining, constrictions or breaks when chromosomes are exposed to partial DNA replication inhibition. They constitute areas of chromatin that fail to compact during mitosis. They are classified as rare or common depending on their frequency within the population and are further subdivided on the basis of their specific induction chemistry into different groups differentiated as folate sensitive or non-folate sensitive rare fragile sites, and as aphidicolin, bromodeoxyuridine (BrdU) or 5-azacytidine inducible common fragile sites. Most of the known inducers of fragility share in common their potentiality to inhibit the elongation of DNA replication, particularly at fragile site loci. There are seven non-similar folate sensitive (FRA10A, FRA11B, FRA12A, FRA16A, FRAXA, FRAXE and FRAXF) and two non-folate sensitive (FRA10B and FRA16B) and have been molecularly characterized. Due to dynamic mutation formed DNA repeat sequences and involving the normally occurring polymorphic CCG/CGG trinucleotide repeats at the folate sensitive and AT-rich minisatellite repeats at the non-folate sensitive fragile sites. These rare fragile site FRAXA is the most important fragile site as it is associated with the fragile X syndrome significant to the clinical side and also explained the predisposition of tumors.

Reference Id: PHARMATUTOR-ART-1196

Introduction:
Establishment of medical genetic makes the possibility to characterized by nonrandom chromosomal abnormality of human different tumor and play an essential role in both clinical diagnosis and basic research with the development of sophisticated instruments with advance knowledge. Human cells can acquire DNA damage through various extrinsic or intrinsic factors. DNA damage commonly arises following exposure to exogenous factors such as UV radiation, ionizing radiation, chemotherapy, and endogenous factors such as reactive oxygen species (Jackson, S. P.,2002) and biological agents leadings to the disturbance of normal physiology of cell during cellular differentiation, providing genomic instability in chromosome. Due to environmental fluctuations, genomic instability increased mutation rate of chromosomal fragility including structural and numerical changes leads to disrupt both transcription and gene expression. Earlier studies have suggested that such type of alteration in genetic material are responsible for the fabrication of fragile sites in cells.Chromosomal fragile sites are defined as regions of the genome which exhibit gaps or breaks on metaphase chromosomes under conditions of partial replication stress (Richards, R. I (2001),Sutherland, G.R. (1991) and Schwartz, M., Zlotorynski, E. and Kerem, B. 2005)

Fragile sites showing a causative relationship and their association with different human genetic disorders such as mental retardation, cancer (ovarian, lung, breast, retinoblastoma and nuroblastoma) are varies between populations. In human genome more than 120 fragile site have been identified (lukusa, T. fryn et. al. 2008.). Fragile site expression does not always appear accompanied by DNA strand breakage and therefore seem to predispose during recombination particularly implicate at breakage-fusion- bridge cycle.

Types of fragile site:
Chromosomal fragile sites are classified according to the chromosome number and officially symbolized by HUGO nomenclature committee in order of acceptance for example the fra(X)(q27.3) site was called FRAXA (fragile site, X chromosome, A site), because this was the ?rst fragile site detected in the 1965 on the X chromosome. According to their frequency in the population and the chemistry of induction, fragile sites are classi?ed as either Rare or Common . Common fragile sites are considered present in all individuals, whereas rare fragile sites are present in a >5% (Glover et al., 1984) small portion of the population with a maximal frequency of 1/20 and 30 rare and 89 common fragile sites have been described (Schwartz, M, .et, al ,2005., Kooy, R.F.et, al 2003 and Sutherland, G.R. (2003).Fragile sites in each category are further classified according to their specific mode of induction in vitro, i.e. their culture requirements (G.R. Sutherland, F. Hech (2003) and (1995).

There are seven rare fragile sites have been characterized at the molecular and clone level: ?ve folate sensitive fragile sites were cloned and found to consist of expanded tandem CGG micro satellite repeats (200 copies) (Jones et al., 1995 and Sutherland, G. R., and R. I. Richards. 1995). These repeats are capable of adopting unusual, non-B DNA structures, such as hairpins (Gacy et al., 1995), slipped strand (S)-DNA (Pearson et al., 1998), or quadruplex DNA (Fry and Loeb, 1994). These various DNA secondary structures can perturb the elongation of DNA replication in vitro andin vivo (Usdin and Woodford, 1995; Samadashwily et al., 1997). And two non-folate-sensitive rare fragile sites were cloned as FRA16B and FRA10B, induced by distamycin A and/or bromodeoxyuridine (BrdU) (Yu, S., M. Mangelsdorf et al., 1997). They are comprised of polymorphic AT-rich minisatellite repeats, and their expression is associated with expansion of one or more of the repeats, up to several kilobases. The expanded FRA16B and FRA10B repeats are highly similar and contain inverted repeats able to form hairpin structures (Handt et al., 2000).

According to records, there are six common fragile sites have been cloned and characterized: FRA3B (Becker, N. A et al.,2002, Pearson, C. E., and R. R. Sinden. 1996, Rassool 1996, Wilke, C. M., S. W. Guo, et al.,1994, Wilke, C. M., B. K. Hall, A. Hoge, et al.,1996 and Zimonjic, D. B., et al., 1997), FRA7G (Hellman, A., et al.,1997 and Huang, H.,et al., 1998), FRA7H (Mishmar, D. et al.,1998), FRA16D (Ried, K et al., 2000 and Mimori, K et al.,1999), FRAXB (Arlt, M. F., et al., 2002), and FRA6F (Morelli, C., et al.,2002). The cytogenetic expression (gaps and constrictions) of these sites is visible along large genomic regions spanning hundreds to thousands of kilobases. Replication time revealed a perturbed elongation of DNA replication along common fragile regions (Wang, L., et al.,1999.). CFS are associated with so many different types of gene .

In mammalian chromosomal organization are divided into two regions differ in their structure and function on the basis of banding pattern. Generally G-bands are AT-rich regions and undergo DNA replication late in the S phase of the cell – cycle while R-bands are GC-rich and DNA replication occurs early inthe S phase (Gardiner, K. 1995). This banding pattern (G- & R-bands) also differ at the organization level and localization of replication loci during condensation of chromatin (Ferreira, J., G. Paolella, C. Ramos, and A. I. Lamond. 1997 and Sadoni, N., S. Langer, C. Fauth, G. Bernardi, T. Cremer, B. M. Turner, and D. Zink. 1999,Gardiner, K. 1995). Furthermore, the cloned fragile sites are found to be enriched in clusters of sequences with high DNA ?exibility relative to non-fragile sequences from R-bands, resembling the ?exibility of G-bands (Mishmar, D. 1999, Mimori, K., T. Druck et.al1999 ,Mishmar, et.al 1998 and D Morelli, C., E et.al 2002 and Ried, K.,et.al., 2000. ). Thus, DNA ?exibility contribute in the expression of common fragile sites, it was important to analyze the ?exibility of common fragile sites mapped to G-bands. Therefore, fragile site are located on light band and may be associated with active gene. Moreover, they have been to coincide with the localization of many oncogene & of communicate to chromosome break point in different cancer type. In numerous reports fragile sites are heritable on human chromosomes (Dekaban A, 1965). Expression of fragile sites in metaphase chromosomes obtained from folic acid deficient lymphocyte cultures and methotrexate (antagonist of folic acid) also induced some rare fragile sites. Thymidine, glycine, and hypoxanthine are inhibitors of folic acid but only thymidine was found to affect expression of fragile sites (Littlefield Jw, Basilico C, 1966It). Genomic instability is a common cause of chromosomal aberrations in many types of tumor cells. DNA damage arises from the exposure of extrinsic or intrinsic factors such as UV radiation, ionizing radiation, chemotherapy, and endogenous factors such as reactive oxygen species (Jackson, 2002). Keefe et al in 2006 also suggested that Chromosomal fragile sites, especially susceptible to DNA breakage, have also been as contributory to the formation of cancer-specific chromosomal aberrations (O'Keefe, et al.2006).

Fragile site (CFS) in the different tumor:
Cytogenetic study includes both structural and numerical chromosomal abnormalities and characteristic feature of malignant cell. Numerous studies suggested that fragile site is closely associated to malignant cell or proto-oncogene (Yunis JJ, Soreng AL. 1984) and could be used as marker for cancer malignancy. Since the first cancer chromosome defect was discovered nearly 30 years ago, more than 80 different types of human malignancy have been found to have recurrent structural rearrangement is probably due to existence of specific labile gaps on human chromosome termed as fragile sites that are highly sensitive to low folic acid and to diverse mutagens and carcinogens. Cultured blood cells have to exposure one of with Antifolates, fluorodeoxyuridine (FdU), aphidicolin, methotrexate (MTX), agents can yield chromosomal rearrangements because chromosomal fragile sites are highly sensitive to mutagens and carcinogens that are microscopically indistinguishable from those found in cancer cells and that act as marker in the identification of cancer cells. These breakpoints of the cancer disorder produced in vitro occur at the same place where fragile sites have been mapped, suggesting that fragile sites may facilitate such rearrangements. According to molecular biology of chromosomal fragile sites may help establish their role in carcinogenesis including telomere sequences TTAGGG (Musio, A. et al., 1996) and whether they are involved in individual disposition to malignancy. Some studies have been suggested that certain nutritional deficiencies may play a role in DNA instability in vivo and their expression may be increase or decrease with higher age of people.

There are so many genotoxic compounds which induce fragile site in the human cell / cell lines and further, suppressed tumor genes. One of the most important inducer is aphidicolin (APC), is DNA polymerase- α, β and γ inhibitor and form Common Fragile sites (CFS), are speci?c chromosomal areas that are prone to form gaps and breaks when cells are exposed to various stresses that agitate DNA replication. (F. Pichiorri, H. et al.,2008). Meticulously, prolonged exposure with low doses of aphidicolin (APC), induce chromosomal fragmentation, breaks at the CFS (T.W. Glover, et al., 1984) and various expressions in the cancer patients and this variation determine the individual expression for predicting cancer risk. The most common type of rearrangements appears to be one or more intra-locus deletions, as opposed to translocations or other rearrangements. CFS extends over hundreds of kilo bases and commonly contain long stretches of AT rich sequences which favor formation of the secondary structures that delay or block the progression of replication forks (S.G. Durkin, T.W. Glover, 2007) .CFS also harbour a number of tumour suppressor genes that are disrupted in tumour cells, such as FHIT (fragile histidine triad) at FRA3B in 3p14.2 or WWOX at FRA16D in 16q23.3 (M. Ohta et al., 1996 and K. Ried et al., .2000). The involvement of these genes in tumour transformation and the expression of speci?c CFS vary according to the cell type.

Conclusion:
This present review described the function and identity of genes located at the fragile sites remains an riddle. Because the first cancer chromosome defect was discovered nearly 30 years ago, more than 80 different types of human malignancy have been found to have recurrent structural chromosomal rearrangement. However, some classes of fragile sites are inherited and that they may therefore be related to a genetic predisposition to the development of malignant diseases. Chromosomal fragile sites that is highly sensitive to low folic acid and to diverse mutagens and carcinogens. They have different types of weak point and have to contribute in the expression of different gene in the different tumor. Some fragile sites are specific to single type of cancer or other are common to different cancer. Therefore, cytogenetic branch of science play important role in the field of clinic as well as research.

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