Expression of matrix metalloproteinase genes in transformed rat cell lines of high and low metastatic potential.

The enzymes that comprise the family of matrix metalloproteinases (MMPs) share the capacity to degrade extracellular matrix components. A large body of evidence indicates that certain members of this metalloproteinase gene family play critical roles in determining the malignant phenotype of solid tumors. We previously have derived transformed cell lines with vastly different metastatic potentials by transfecting different combinations of oncogenes into primary rat embryo cells. Conditioned medium from those cell lines was assayed by Western blot analysis for the production of four separate matrix metalloproteinases to see whether a correlation could be found between protease expression and the metastatic phenotype. The transformed rat embryo cell lines with high metastatic potential were found to produce high levels of the stromelysin 1 (MMP-3) and stromelysin 2 (MMP-10) proteases, while the nonmetastatic lines produced low or undetectable levels of these two enzymes. No correlation was seen between the metastatic phenotype of the cell lines and the level of expression of two other matrix metalloproteinases, the M(r) 72,000 type IV collagenase (MMP-2) and the M(r) 92,000 gelatinase (MMP-9). These data suggest that the differential regulation of the stromelysin proteases may contribute to the difference seen in the metastatic potential of these cell lines.

Allelic loss on chromosome 8p12-21 in microdissected prostatic intraepithelial neoplasia.

The development and progression of human prostate cancer is associated with genetic abnormalities in tumor cells. Inactivation of tumor suppressor genes due to allelic loss is thought to be an important mechanism of gene alteration in prostatic neoplasms. In this study we examined allelic loss on chromosome 8p12-21 in microdissected samples of normal prostatic epithelium, high grade prostatic intraepithelial neoplasia (PIN), and invasive prostate carcinoma from the same patients. Tissue microdissection under direct microscopic visualization procures pure populations of cells of interest, including small lesions such as PIN. Among 30 patients with concomitant cancer and PIN, we found loss of heterozygosity on chromosome 8p12-21 in 63% (34 of 54) of foci of PIN examined and 90.6% (29 of 32) of tumors, suggesting that abnormalities on chromosome 8p12-21 may be important in the early stages of prostatic carcinoma development. Several cases in which multiple foci of PIN from the same patient were sampled showed different patterns of allelic loss. Fifty-five % (16 of 29) of the prostate carcinomas contained a potential precursor PIN focus based on allelic loss pattern. Our results are consistent with the hypothesis that PIN arises multifocally within the prostate gland, and that a subset of these lesions progress to become carcinoma.

Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12-21.

To investigate the possible involvement of a tumor suppressor gene(s) on chromosome 8 in prostatic neoplasms, we performed a comprehensive loss of heterozygosity (LOH) study on 99 tumors from 97 prostate cancer patients. One of the carcinomas was a lymph node metastasis; the other 98 were primary carcinomas. Pure populations of carcinoma cells and normal epithelia were procured by tissue microdissection. Two separate tumor foci were obtained from each of two patients. Microsatellite markers from 25 loci on the short arm and one locus on the long arm of chromosome 8 were used for PCR-based LOH analysis on matched normal and tumor DNA samples. The overall LOH on 8p in this study was 85.9% (85 of 99) of carcinomas. The loss was highest at markers D8S133, D8S136, NEFL, and D8S137 (62,72, 64, and 75%, respectively), which are located at 8p12-21. Seventy-nine of 99 tumors exhibited loss in at least one of these four loci. In contrast, LOH at 8p22 was much lower: 17,18,18, and 19% at D8S549, D8S602, D8S254, and D8S261, respectively, with 25 of 99 tumors showing deletion in one or more of the four loci. All but 5 tumors with deletions in this more distal region had at least one retained locus between the 8p22 deletion and a more proximal region of loss at 8p12-21; 1 tumor had loss at 8p22 but not 8p12-21. This suggests there may be two distinct regions of loss and, therefore, two tumor suppressor genes on this chromosomal arm. The loss on 8p12-21 showed little or no correlation with grade or stage of disease.

Purine mutants of mammalian cell lines: III. Control of purine biosynthesis in adenine phosphoribosyl transferase mutants of CHO cells.

Spontaneous and mutagen-induced 2,6-diaminopurine-resistant mutants of Chinese hamster ovary (CHO-K1) cells were isolated. Such mutants fell into two classes: spontaneous and ethylmethane-sulfonate-induced mutants had approximately 5% wild-type adenine phosphoribosyl transferase (APRT) activity, whereas ICR-170G-induced mutants had barely detectable APRT activity. Since it has been reported that human hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Lesch-Nyhan syndrome) and APRT mutants over-produce purines, we examined the control and rate of purine biosynthesis in the Chinese hamster mutants. End product inhibition by adenine could not be demonstrated in such mutants, indicating that the active feedback inhibitor is a nucleotide rather than the free purine base, HGPRT activity was normal in all mutants examined except in one isolate. Purine biosynthesis as measured by the accumulation of the purine biosynthetic intermediate phosphoribosyl formylglycineamide was not elevated in the mutants as might have been predicted from work with Lesch-Nyhan cells. The data also suggest that our strain of CHO-K1 is physically or functionally haploid for the APRT locus.

Chromosome 16 allelic loss analysis of a large set of microdissected prostate carcinomas.

PURPOSE: To perform loss of heterozygosity (LOH) analysis on chromosome 16 in 102 highly purified DNA samples isolated from one or more adenocarcinomas, prostatic intraepithelial neoplasia (PIN), and matched benign prostatic epithelium from 95 radical prostatectomy patients. MATERIALS AND METHODS: Specimens were procured by microdissection of frozen tissue samples, thus ensuring that highly select pure populations of cells were obtained for DNA extraction and LOH analysis. Multiple microsatellite markers were used to determine allelic loss on chromosome 16q. RESULTS: Overall loss on 16q was seen in 31% of the cancers, and occurred more frequently in high stage cancers than low stage cancers. In contrast, allelic loss in PIN failed to exceed 6% at any of the loci that were examined. CONCLUSIONS: These results suggest that inactivation of a putative tumor suppressor gene on 16q may be involved in the progression of some prostate cancers.