Inverse association of p16 INK4a and p14 ARF methylation of the CDKN2a locus in different Gleason scores of prostate cancer.

BACKGROUND: Promoter hypermethylation is an important epigenetic mechanism in the regulation of several key modulators of prostate carcinoma progression. Recent studies suggest that the polycomb-group (PcG) protein BMI1 may have an impact on epigenetic regulation of several targets, including the CDKN2a locus. METHODS: In this study, we investigated the association of BMI1 expression, promoter methylation of CDKN2a (p16(INK4a) and p14(ARF)) and TMS1 with pathological variables (Gleason score, TNM stage, perineural invasion) in prostate cancer (PCa). RESULTS: Methylation of p16(INK4a) and p14(ARF) revealed an inverse association with Gleason score 7b and Gleason score 6. No significant association could be demonstrated for BMI1 -overexpression and promoter methylation of p16(INK4a), p14(ARF) and TMS1 as well as pT category. CONCLUSIONS: Our data suggest that the CDKN2a locus is a switch in PCa with methylation of p16(INK4a) being a marker for more aggressive tumours of Gleason score 7b, but no association with BMI overexpression was observed.

The G2/M regulator 14-3-3sigma prevents apoptosis through sequestration of Bax.

In response to DNA damage and genotoxic stress, the p53 tumor suppressor triggers either cell cycle arrest or apoptosis. The G(2) arrest after damage is, in part, mediated by the p53 target, 14-3-3final sigma (final sigma). Colorectal tumor cells lacking final sigma are exquisitely sensitive to DNA damage. Here we analyzed the mechanism of this sensitivity in final sigma(-/-) as compared with final sigma(+/+) human colorectal tumor cells. Exposure to adriamycin resulted in rapid apoptosis only in final sigma(-/-) cells. This was further characterized by caspase-3 activation, p21(CIP1) cleavage, and CDK2 activation. Moreover, Bax was rapidly translocated out of the cytoplasm, and cytochrome c was released in final sigma(-/-) cells. Transient adenovirus-mediated reconstitution of final sigma in the final sigma(-/-) cells led to effective rescue of this phenotype and protected cells against apoptosis. The association of final sigma, Bax, and CDK1 in protein complexes may be the basis for this antiapoptotic mechanism. In conclusion, final sigma not only enforces the p53-dependent G(2) arrest but also delays the apoptotic signal transduction.

Induction of polyploidy and apoptosis after exposure to high concentrations of the spindle poison nocodazole.

The proportions of aneuploid/polyploid versus euploid cells formed after treatment with spindle poisons like nocodazole are of course dependent on the relative survival of cells with numerical chromosome aberrations. This work aimed at studying the survival of polyploid cells formed after treatment with a nocodazole concentration sufficient to significantly decrease tubulin polymerization (0.1 microg/ml). First, normal primary lymphocytes were analysed and the following complementary chromosomal parameters were quantified: mitotic index, frequency of abnormal mitoses, polyploid metaphases and apoptotic cells. The results clearly indicate a positive correlation between abnormal mitotic figures, apoptosis and the induction of polyploidy. They therefore led to a single cell approach in which both apoptosis and polyploidy induction could be scored in the same cell. For this purpose, actively proliferating cells are required and two human leukaemic cell lines were used, KS (p53-positive) and K562 (p53-negative), which have a near-triploid karyotype. Cells were separated into an apoptotic and a viable fraction by means of annexin-V staining and flow cytometry. In KS, treatment with nocodazole induced a similar fraction of hexaploid cells in both the viable and apoptotic fraction, but no dodecaploid cells were ever observed. In contrast, a population of dodecaploid cells (essentially viable) was clearly observed in the K562 cell line. The results in KS, as compared with K562, confirm that wild-type p53 can prevent further cycling of polyploid cells by blocking rereplication. The most probable explanation for these data is that not only the mitotic spindle but also interphase microtubules are sensitive to nocodazole treatment. Our data thus strongly suggest that besides the G(1)/S checkpoint under the control of p53, the G(2)/M transition may be sensitive to depolymerization of microtubules, possibly under the control of Cdc2, Bcl-2, Raf-1 and/or Rho.

Towards a unifying model for the metaphase/anaphase transition.

The term mitosis actually covers a complex sequence of events at the level of the cell membrane, the cytoplasm, the nuclear membrane and the chromosomes; recently attention has been focused more and more on the checkpoints that control their orderly progression. The term 'checkpoint' refers here to the inhibitory pathways that coordinate coupling between the sequence of events, ensuring dependence of the initiation of each upon successful completion of others. This paper will mainly focus upon the possible checkpoint which controls a brief but essential step, dissociation of the sister chromatids into two identical chromosomes. This step will be called the metaphase/anaphase transition. First, the molecular components that are important in metaphase/anaphase transition will be reviewed: accurate segregation of sister chromatids between the daughter cells is dependent on coordinated interaction of centrosomes, centromeres, kinetochores, spindle fibres, topoisomerases, proteolytic processes and motor proteins. Deficiencies in or impairment of any of these structures or in their control systems may lead to a more or less important genomic imbalance. A model combining the ultrastructural components, the molecular components and the controlling molecules will be proposed. The unifying concept emerging from this synthesis indicates that sister chromatids separate independently of the tubulin fibres, as a result of proteolytic processes controlled by the anaphase promoting complex. The spindle fibres are thus necessary to move the separated chromatids to the spindle poles but probably not to initiate separation. A number of remaining questions are also highlighted.

Numerical aberrations of chromosomes 1 and 17 in tumor cell lines of the exocrine pancreas as determined by fluorescence in situ hybridization.

The relationship between the copy numbers of chromosomes 1 and 17 and characteristics related to aggressiveness, histological grade, and doubling time was studied in 10 cell lines from pancreatic carcinomas (7 from primary tumors, 2 from ascites, and 1 from a liver metastasis). Fluorescence in situ hybridization with, respectively, the (peri-)centromeric pUC 1.77 (chromosome 1), and D17Z1 (chromosome 17) probes was applied to interphase nuclei. The results showed that most of these cell lines were hyperploid for both chromosomes studied in accordance with their chromosome counts in metaphase; moreover, there existed a statistically significant positive correlation between the results for both probes. Two of these cell lines had a higher mean copy number for chromosome 17 than for chromosome 1 (PaCa 44 and PaTu 2); two of them had a higher mean copy number for chromosome 1 than for chromosome 17 (Panc 1 and PSN 1). Although only a weak correlation was observed between the number of signals for either chromosomes 1 or 17 and the doubling time, lines of grade 1 and 2 showed a lower average number of spots per nucleus than grade 3 cell lines for both chromosomes studied.

Improved detection of DNA aneuploidy in primary breast cancer using quantitative DNA image analysis in combination with fluorescent in situ hybridization technique.

To obtain more information about the relationship between numerical aberrations of chromosome 1 and the overall DNA content of breast cancer cells, fluorescent in situ hybridization with a pericentromeric probe for this chromosome and image analysis based densitometry were carried out on imprints of benign (15 cases, mainly fibroadenomas) and malignant breast disease (31 invasive ductal carcinomas out of 45 cases). The most pronounced aneuploidy was observed in invasive ductal and lobular carcinoma cases both by in situ hybridization and DNA content (76.7 and 75.0% were aneuploid). The frequency of cells with two spots for chromosome 1 was 48.3 and 51.5%, respectively, as compared to 80.3% in control lymphocytes. There was a weak overall correlation (r2 = 0.83) between DNA content and copy number of chromosome 1 in the malignant samples, although some of the DNA diploid/near diploid carcinomas showed a marked aneusomy for this chromosome. Also, some aberrations were present in the benign breast disease samples. Classification of cases by a linear discriminant analysis was most accurate when both techniques were combined (77% of cases correctly classified, according to anatomo-pathological diagnosis). The variables which received the highest weight in the linear discriminant function are the percentage DNA-diploid cells and the fraction of cells with two spots for chromosome 1. The sensitivity and sources of error of both techniques is considered.

Aneuploidy for chromosome 1 and overall DNA content in benign and malignant breast disease.

Fluorescence in situ hybridization (FISH) with a probe for the pericentromeric region of chromosome 1 and DNA content measurements by image-analysis-based densitometry have been carried out on imprints of benign and malignant breast tissue. In general, an increase in the number of spots per nucleus was observed in the invasive carcinomas, with a large intercellular variation. In comparison with lymphocytes from controls, some cases of benign breast disease already had an increased frequency of aneusomy of chromosome 1, although they were all (near)diploid by DNA-content. However, an overall concordance between the DNA content measurements and the results of FISH was observed, although some exceptions were seen. A statistically significant correlation between the DNA index and the mean number of spots for chromosome 1 per nucleus was found. A linear discriminant analysis was applied on the data; the resulting classification of patients was most accurate when parameters describing DNA content and FISH results were combined.

Patterns of interallelic divergence at the rabbit b-locus of the immunoglobulin light chain constant region are in agreement with population genetical evidence for overdominant selection.

Population studies at the b-locus of the "constant" regions of the rabbit immunoglobulin kappa 1 light chain (c kappa 1) revealed patterns of gene diversity resembling those that mark the peculiar nature of the major histocompatibility complex, such as large number of alleles, high heterozygosity levels, consistent excess of heterozygous individuals and long allele coalescence times. This paper documents the evolutionary patterns at the b-locus as inferred from DNA sequence comparisons. Among alleles, synonymous substitutions outnumbered expectations for neutral alleles by an order of magnitude. They were distributed randomly throughout the c kappa 1 coding region while interallelic amino acid differences did cluster into segments overlapping with the regions exposed to the solvent. Within these regions, acceptance rates of mutation at amino acid replacement sites were even higher than those at synonymous sites (dr/ds = 1.6-3.0), while in the intervals between these regions the opposite was found (dr/ds approximately 0.3). Under the assumption that allelic variation is adaptive at the molecular surface, the divergence patterns at the b-locus are therefore very similar to those reported for the major histocompatibility complex. An analysis at the quasi silent bas-locus (c kappa 2), which is linked to the b-locus, and comparisons among genes of the "variable" region of the kappa 1 light chains (v kappa 1), revealed patterns of divergence which differed markedly from those observed at the c kappa 1 constant regions. It is suggested that allelic variability at immunoglobulin constant regions can be due to mechanisms similar to those enhancing diversity at histocompatibility loci.