Molecular and fluorescence in situ hybridization characterization of the breakpoints in 46 large supernumerary marker 15 chromosomes reveals an unexpected level of complexity.

Supernumerary marker chromosomes (SMCs) of chromosome 15, designated "SMC(15)s," are the most common SMC in humans, accounting for as much as 60% of all those observed. We report the characterization of 46 large SMC(15)s, using both fluorescence in situ hybridization and polymerase chain reaction analysis within and distal to the Prader-Willi/Angelman syndrome critical region (PWACR). Our aim was to establish detailed information on origin, content, and breakpoints, to address the formation of SMC(15)s, and to facilitate genotype-phenotype correlations. For all patients in whom we were able to establish the parental origin, the SMC(15)s were maternally derived. Two patients were observed who had familial SMC(15)s, both inherited from the mother; however, in all remaining patients for whom parental samples were available, the SMC(15)s were shown to have arisen de novo. With one exception, all the SMC(15)s were shown to include the entire PWACR. Detailed investigations of the distal breakpoints categorized the SMC(15)s into two groups. Group A, representing approximately two-thirds of the SMC(15)s, had a breakpoint beyond the standard distal PWS/AS deletion breakpoint BP3, at a position close to the microsatellite marker D15S1010 and the bacterial artificial chromosome 10I10. The group B SMC(15)s were shorter, with more variable breakpoints located around BP3. The majority of the SMC(15)s were shown to have asymmetrical breakpoints, with the two inverted arms of the SMC being unequal in length. Our study revealed an unexpected level of complexity and heterogeneity among SMC(15)s that is not seen in other chromosome 15 rearrangements, such as deletions and duplications. This suggests that multiple mechanisms are involved in the formation of large SMC(15)s.

Genomic instability-based transgenic models of prostate cancer.

To develop animal models that represent the broad spectrum of human prostate cancer, we created transgenic mice with targeted prostate-specific expression of two genes (ECO:RI and c-fos) implicated in the induction of genomic instability. Expression of the transgenes was restricted to prostate epithelial cells by coupling them to the tissue-specific, hormonally regulated probasin promoter (PB). The effects of transgene expression were examined histologically in prostate sections at time points taken from 4 to 24 months of age. The progressive presence of regions of mild-to-severe hyperplasia, low- and high-grade prostatic intra-epithelial neoplasia, and well-differentiated adenocarcinoma was observed in both PBECO:RI lines but no significant pathology was detected in the PBfos line. Prostate tissue of PBECO:RI mice was examined for expression of p53, proliferating cell nuclear antigen (PCNA) and Ki67 at multiple time points. Although p53 does not appear to be mutated, levels of PCNA and Ki67 are elevated and correlate with the severity of the prostatic lesions. Overall, pre-neoplastic and neoplastic stages represented in the PBECO:RI model showed similarity to corresponding early stages of the human disease. This genomic instability-based model will be used to study the mechanisms involved in the early stages of prostate carcinogenesis and to investigate the nature of subsequent events necessary for the progression to advanced disease.

Design and testing of ribozymes for cancer gene therapy.

This chapter describes procedural aspects for development of ribozymes in general, and specifically, that cleave mRNA to an essential cellular gene, the AC40 subunit of RNA pol I. Ribozyme design includes functional selection of binding sites followed by computer modeling. These ribozymes are being used in vectors that target expression to the prostate via tissue specific promoters (Voeks, Norris, and Clawson, 1998) and have demonstrated efficacy.

Overexpression and amplification of c-myc in the Syrian hamster kidney during estrogen carcinogenesis: a probable critical role in neoplastic transformation.

An estrogen receptor-driven, multistep process for estrogen carcinogenesis in the Syrian hamster kidney is proposed. Because in this species the reproductive and urogenital tracts arise from the same embryonic germinal ridge, it is evident that the kidney has carried over genes that are responsive to estrogens. Using in situ hybridization, overexpression of early estrogen-response genes, i.e., c-myc and c-fos, has been shown to be localized preferentially in early renal tumor foci after 3.5-4.0 months of estrogen treatment. This event coincides with an increased number of S-phase proliferating cell nuclear antigen-labeled cells in these tumor foci, along with a rapid rise in aneuploid frequency in the kidney. Western blot analyses of c-MYC and c-FOS protein products support the overexpression of these genes. Amplification of c-myc, 2.4-3.6-fold, but not of c-fos, was detected in 67% of the primary renal tumors examined, by Southern blot analyses. Consistent chromosomal gains, common to both diethylstilbestrol- and estradiol-induced renal neoplasms, were observed in chromosomes 1, 2, 3, (6), 11, (13), 16, 20, and 21 (chromosome number alterations are indicated in parentheses). Using fluorescence in situ hybridization, the c-myc gene was localized to hamster chromosome 6qb. Chromosome 6 exhibited a high frequency of trisomies and tetrasomies in the kidney after 5.0 months of estrogen treatment and in primary renal tumors. The data presented indicate that estrogen-induced genomic instability may be a key element in carcinogenic processes induced by estrogens.

Glutathione S-transferase Yc cDNA from Syrian hamster kidney.

A cDNA encoding alpha-class glutathione S-transferase Yc (GSTYc) has been isolated from a Syrian hamster kidney library, and its nucleotide sequence (968 bp) has been determined. Analysis of the deduced amino acid sequence revealed a high level of identity between Syrian hamster GSTYc, rat GST Yc1 and Yc2 and mouse GSTYc. Northern-blot experiments demonstrated that Syrian hamster GSTYc expression is tissue-specific. A GSTYc mRNA of approx. 1 kb is expressed in liver, kidney, vas deferens and epididymis. Expression of the GSTYc transcript was not detected in testis or uterus.