Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.     

Heterochromatin variation and sex chromosome polymorphism in Nesokia indica: a population study

Nesokia indica, the Indian mole rat, exhibits extensive variability (polymorphism) for the constitutive heterochromatin of the X and Y chromosomes. These polymorphic X and Y types range from a large metacentric chromosome to a small acrocentric one and occur in different frequencies in the population. On the assumption that there is random mating among individuals carrying these various X and Y chromosomes, the population shows Hardy-Weinberg proportions for the genotypes. However, notwithstanding the partial or total loss of constitutive heterochromatin of the X and Y chromosomes in a few individuals, its retention in most of the animals seems obligatory to the population at large. Hence, we suggest that the C-heterochromatin plays a "regulatory" role in the population dynamics of this species.     

Genetically determined asynapsis, spermatogenic degeneration, and infertility in men.

We report a family in which azoospermia and infertility affected two sibs whose parents were first cousins once removed. Meiotic cells of the proband, who had the chromosomal complement of a normal male (46,XY), exhibited asynapsis, defective synaptonemal complex (SC) formation, chiasma failure, and degeneration of prophase spermatocytes with asynapsis. Based on these observations, we suggest that the meiotic abnormalities and infertility in this family comprise a trait with an autosomal recessive mode of inheritance. Review of published cases of infertile men with normal chromosomal complements and disturbed meiosis suggests that genetically determined asynapsis and desynapsis similar to that established in plant and insect species also occur in humans. In humans, asynapsis appears to be inherited as an autosomal recessive. The mode of inheritance of desynapsis is not clear; X-linked recessive or autosomal dominant has been suggested in one family. Studies by us and by others reported in the literature suggest that the mode of action of genes that affect synapsis and cause a reduction in the numbers of visible chiasmata at diakinesis is dissimilar to that of the action of genes that cause defective meiotic recombination, defective repair of induced damage to DNA in somatic cells, and chromosome instability.     

Identification of promoters bound by c-Jun/ATF2 during rapid large-scale gene activation following genotoxic stress

The NH2-terminal Jun kinases (JNKs) function in diverse roles through phosphorylation and activation of AP-1 components including ATF2 and c-Jun. However, the genes that mediate these processes are poorly understood. A model phenotype characterized by rapid activation of Jun kinase and enhanced DNA repair following cisplatin treatment was examined using chromatin immunoprecipitation with antibodies against ATF2 and c-Jun or their phosphorylated forms and hybridization to promoter arrays. Following genotoxic stress, we identified 269 genes whose promoters are bound upon phosphorylation of ATF2 and c-Jun. Binding did not occur following treatment with transplatin or the JNK inhibitor SP600125 or JNK-specific siRNA. Of 89 known DNA repair genes represented on the array, 23 are specifically activated by cisplatin treatment within 3-6 hr. Thus, the genotoxic stress response occurs at least partly via activation of ATF2 and c-Jun, leading to large-scale coordinate gene expression dominated by genes of DNA repair.     

“Down syndrome: an insight of the disease”

Down syndrome (DS) is one of the commonest disorders with huge medical and social cost. DS is associated with number of phenotypes including congenital heart defects, leukemia, Alzeihmer’s disease, Hirschsprung disease etc. DS individuals are affected by these phenotypes to a variable extent thus understanding the cause of this variation is a key challenge. In the present review article, we emphasize an overview of DS, DS-associated phenotypes diagnosis and management of the disease. The genes or miRNA involved in Down syndrome associated Alzheimer’s disease, congenital heart defects (AVSD), leukemia including AMKL and ALL, hypertension and Hirschprung disease are discussed in this article. Moreover, we have also reviewed various prenatal diagnostic method from karyotyping to rapid molecular methods -  MLPA, FISH, QF-PCR, PSQ, NGS and noninvasive prenatal diagnosis in detail.     

The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression

Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G1 phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.     

STR markers for detecting heterogeneity in Indian population

Short tandem repeats are highly polymorphic sequences of nucleotides, which are abundant in eukaryotic genome. They form approximately 3% of the total human genome and occur on average in every 10, 000 nucleotides. Due to their small dimension, low mutation, and high level of polymorphism, these markers are intensely used as important genetic markers for mapping studies, disease diagnosis, and human identity testing. In the present study allelic distribution of four autosomal short tandem repeat markers (D21S2055, D21S11, D21S1435 and D21S1411) has been analyzed in Indian population. For determination of heterogeneity and their allelic frequency QF-PCR analysis have been done. All the loci were found highly polymorphic. Marker D21S1411 was the most informative (93.6%) and D21S1435 (70.1%) was the least informative marker in Indian population.     

EST-derived genic molecular markers: development and utilization for generating an advanced transcript map of chickpea

Well-saturated linkage maps especially those based on expressed sequence tag (EST)-derived genic molecular markers (GMMs) are a pre-requisite for molecular breeding. This is especially true in important legumes such as chickpea where few simple sequence repeats (SSR) and even fewer GMM-based maps have been developed. Therefore, in this study, 2,496 ESTs were generated from chickpea seeds and utilized for the development of 487 novel EST-derived functional markers which included 125 EST-SSRs, 151 intron targeted primers (ITPs), 109 expressed sequence tag polymorphisms (ESTPs), and 102 single nucleotide polymorphisms (SNPs). Whereas EST-SSRs, ITPs, and ESTPs were developed by in silico analysis of the developed EST sequences, SNPs were identified by allele resequencing and their genotyping was performed using the Illumina GoldenGate Assay. Parental polymorphism was analyzed between C. arietinum ICC4958 and C. reticulatum PI489777, parents of the reference chickpea mapping population, using a total of 872 markers: 487 new gene-based markers developed in this study along with 385 previously published markers, of which 318 (36.5%) were found to be polymorphic and were used for genotyping. The genotypic data were integrated with the previously published data of 108 markers and an advanced linkage map was generated that contained 406 loci distributed on eight linkage groups that spanned 1,497.7 cM. The average marker density was 3.68 cM and the average number of markers per LG was 50.8. Among the mapped markers, 303 new genomic locations were defined that included 177 gene-based and 126 gSSRs (genomic SSRs) thereby producing the most advanced gene-rich map of chickpea solely based on co-dominant markers.