Use of PCR amplification of cDNA to study mechanisms of human cell mutagenesis and malignant transformation.

PCR is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. But some investigators need to sequence the entire coding region of mammalian genes to determine what specific changes have occurred. In 1989, we [Yang et al: Gene 83:347-354] described a method to copy mRNA of the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene directly from the lysate of a clone of 6-thioguanine-resistant mutant diploid human fibroblasts without the need for RNA extraction or DNA template purification. To avoid detecting random changes introduced by polymerases, 100 to 500 cells from an individual clone, each containing the identical mutation, are lysed and the cDNA is amplified 10(10)-to 10(11)-fold to obtain 5 to 10 micrograms of DNA. The consensus sequence of the cDNA is determined by direct nucleotide sequencing. Using this method, we have investigated the kinds of mutations induced by carcinogens in the coding region of the HPRT gene and their location in the gene and examined the role of DNA repair in this process. Normal repair-proficient human cells and cells deficient in DNA repair were exposed to mutagens in exponential growth or synchronized and exposed at the beginning of S phase or in G1 phase several hr prior to DNA replication. The kinds and location of mutations in the HPRT gene were determined and knowledge of the nature of the DNA lesions formed by the various mutagens allowed assignment of the DNA strand in which the premutagenic lesion that gave rise to the mutation had been located. Related assays involving PCR have been used to determine the nature of mutations in the coding region of the H-, N-, or K-ras genes of tumor-derived malignant human cells and to determine whether or not such cells express specific growth factor genes.

Hirudin insensitive thrombin-stimulated platelet release.

It has been previously proposed that platelet stimulation may involve two platelet-thrombin complexes: an initial platelet-thrombin complex (P-T) which is converted to an activated platelet-thrombin complex (P*-T). By using the release of radioactive serotonin as a measure of thrombin stimulation, we have demonstrated that under appropriate conditions, a hirudin sensitive and a hirudin insensitive complex can be differentiated. At short platelet-thrombin preincubation times (0-2 minutes) at 4 degrees C, in a buffer containing 18.7 mM phosphate, added hirudin almost completely inhibited the release of radioactive serotonin obtained upon subsequent warming to 37 degrees C (only the hirudin sensitive complex exists). If platelets were preincubated with thrombin for longer periods of time (30 minutes), hirudin became less effective in inhibiting the release obtained upon subsequent warming to 37 degrees C. The same results were obtained whether or not the platelets were washed after incubation at 4 degrees C and before warming to 37 degrees C. We postulate that this change in hirudin sensitivity may reflect a slow conversion of the first platelet-thrombin complex (P-T) to an activated platelet-thrombin complex (P*-T) which can undergo release upon warming. This transition appears to be much faster in acetatetris buffer since at short platelet-thrombin incubation times at 4 degrees C, added hirudin had little or no effect on the release obtained upon warming to 37 degrees C. The difference in the ability of hirudin to inhibit thrombin-induced release in the two buffers was shown to depend upon the presence of phosphate and on variations in ionic strength, and not due to a change in the inhibition constant (Ki) for hirudin.

Malignant transformation of human fibroblasts by ionizing radiation.

As one step in developing an assay for quantifying the induction of malignant transformation of human cells by ionizing radiation, we exposed cells from a non-tumorigenic, infinite life span, near-diploid fibroblast strain MSU-1.1 to 4.35 Gy 60Co radiation and assayed them for focus formation. The mean frequency of foci in the irradiated population was 6 x 10(-7) cells assayed. No foci were found in the control cells. Of four focus-derived cell strains studied in detail, two produced malignant tumours within 3-7 weeks. The other two did not produce tumours during the 12-month period of study. The tumours from one strain were classified as sarcomas composed exclusively of spindle-shaped cells. Tumours from the other strain were sarcomas consisting of a mixed population of round and spindle cells. Immunoprecipitation analysis of the status of the p53 gene in the focus-derived strains, using a mutant-specific anti-body (Pab240) and an antibody that recognizes both mutant and wild-type p53 protein (Pab421), showed that the tumorigenic strains were completely devoid of p53 protein. One non-tumorigenic strain expressed wild-type p53 protein, and the other expressed a lower molecular weight form of the protein. Karyotypic analysis showed that the tumour-derived cells from one tumorigenic strain had lost one copy of chromosome 6, 14, 16 and 17. The tumour-derived cells from the second strain had lost one copy of chromosome 7, 13, 14 and 17 and part of chromosome 6, as well as part of the other copy of chromosome 7 and 17. These results suggest that the common loss of one copy of chromosome 14, 17 and part of 6 plays a causal role in the malignant transformation of these cells. Furthermore, the results indicate that it will be possible to develop a system that uses near-diploid human fibroblasts to quantify radiation-induced malignant transformation.

The lack of a role for protein kinase C in neurite extension and in the induction of ornithine decarboxylase by nerve growth factor in PC12 cells.

Nerve growth factor (NGF) causes pheochromocytoma cells (PC12) to undergo a number of physiological changes which mimic the differentiated neuronal cell, including neurite extension. We have examined protein kinase C (Ca2+/phospholipid-dependent enzyme) as a potential signaling mechanism in NGF-stimulated neurite outgrowth and induction of the enzyme ornithine decarboxylase. Phorbol 12-myristate 13-acetate (PMA) can activate protein kinase C and induce ornithine decarboxylase in PC12 cells with kinetics which are similar to those of NGF induction, but only to levels about 10-fold lower. The induction of ornithine decarboxylase by both NGF and PMA is inhibited by cycloheximide and actinomycin D suggesting that both agents increase enzyme activity by increasing gene transcription. The evidence presented here, however, shows that the induction produced by the two agents is through two different pathways. First, maximal induction by NGF is increased when PMA is included in the media showing that the two effects are synergistic. Second, NGF does not cause induction of ornithine decarboxylase in the mutant PC12nnr5 cell line (Green, S.H., Rydel, R.E., Connolly, J.L., and Greene, L.A. (1986) J. Cell Biol. 103, 1967-1978) while added PMA does produce an induction of the enzyme. Finally, when protein kinase C is down-regulated by incubating PC12 cells with PMA in serum-containing or serum-free medium for 24 h, the induction by PMA is completely inhibited, while the NGF induction is not affected. A recent study (Hall, F.L., Fernyhough, P., Ishii, D.N., and Vulliet, P.R. (1988) J. Biol. Chem. 263, 4460-4466) using sphingosine inhibition concluded that protein kinase C was required for NGF-stimulated neuritogenesis. In contrast, results presented here show that down-regulation of protein kinase C also has no effect on NGF-mediated neurite extension in PC12 cells grown in serum-free medium. Our data demonstrate that induction of ornithine decarboxylase and formation of neurites in PC12 cells by NGF does not require a protein kinase C-mediated pathway.

Molecular staging of colorectal cancer: K-ras mutation analysis of lymph nodes upstages Dukes B patients.

PURPOSE: Multiple attempts have been made to improve the clinical/pathologic staging system of Dukes to focus adjuvant therapy decisions. The purpose of this study was to determine whether K-ras mutational status of regional nodes in patients with Dukes B2 colorectal cancer could be used to stage their disease more accurately. METHODS: Using formalin-fixed, paraffin-embedded archival material, tumor samples were screened for K-ras mutations using a mutation-specific polymerase chain reaction method, followed by gel electrophoresis in a 96-well array. Patients with Dukes B2 tumors that have mutations in codon 12 or 13 of the K-ras gene were identified. RESULTS: Mutational analysis of the lymph nodes from these patients revealed an 80 percent (16/20) incidence of the same mutations in regional lymph nodes. None of the four patients with mutation-free nodes developed recurrence compared with 37.5 percent (6/16) with K-ras positive lymph nodes. CONCLUSIONS: The data suggest that patients with Dukes B2 colorectal cancers that have mutations in codon 12 or 13 of the K-ras gene are at high risk for the development of nodal metastases. Mutational analysis of the lymph nodes identifies high-risk patients who should be considered for adjuvant chemotherapy. Therefore, K-ras mutational analysis should be considered for molecular staging of colorectal cancer.

Malignant transformation of human fibroblast cell strain MSU-1.1 by (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene.

Treatment of MSU-1.1 cells, a near-diploid, karyotypically stable, infinite life-span human fibroblast strain, with (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene induced focus formation. Eight independent foci were isolated and the cell strains developed from them were examined for characteristics of malignant cells. Each grew to a higher density in medium containing 1% serum than did the MSU-1.1 cells. Three of the eight grew rapidly in serum-free medium without added growth factors, formed colonies in agarose with diameters of greater than or equal to 120 microns at a frequency of 5-19%, exhibited loss of genetic material, and, when injected into athymic mice, formed sarcomas that reached 6 mm in diameter within 2-3 wk. One produced high-grade sarcomas (progressively growing, invasive tumors exhibiting high mitotic activity); the other two produced low-grade sarcomas (tumors with a lower degree of mitotic activity) that developed focal areas of high-grade malignant cells if left in the animals for greater than 4 wk. A fourth cell strain formed high-grade sarcomas only after 2.5-3 mo, but the tumor-derived cells analyzed showed the same growth properties as the three malignant cell strains described above, exhibited loss of genetic material, and, when reinjected into athymic mice, produced high-grade sarcomas with a short latency period.

Triiodothyronine-induced accumulations of malic enzyme, fatty acid synthase, acetyl-coenzyme A carboxylase, and their mRNAs are blocked by protein kinase inhibitors. Transcription is the affected step.

Addition of triiodothyronine (T3) to chick-embryo hepatocytes in culture causes increased accumulations of malic enzyme, fatty acid synthase, acetyl-CoA carboxylase and their mRNAs. H-8 and other protein kinase inhibitors inhibited the T3-induced accumulations of these lipogenic enzymes and their mRNAs but had no effect on the activities of 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase, enzymes not induced by T3 in chick-embryo hepatocytes. H-8 also had no effect on the activities of malic enzyme, fatty acid synthase, and acetyl-CoA carboxylase in hepatocytes not treated with T3. Synthesis of soluble protein, levels of mRNAs for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, and induction of metallothionein mRNA by Zn2+ were unaffected by H-8 at concentrations that inhibited the T3-induced accumulation of lipogenic enzymes and their mRNAs. H-8 inhibited T3-induced transcription of the genes for both malic enzyme and fatty acid synthase but had little effect on transcription of the beta-actin or glyceraldehyde-3-phosphate dehydrogenase genes or on total RNA synthesis in isolated nuclei. H-8 also had no effect on binding of T3 to its nuclear receptor. In isolated nuclei, H-8 inhibited phosphorylation of total protein by 15-20%. Phosphorylation of only one major protein was consistently and substantially inhibited, indicating that the effect of H-8 was selective. These results suggest that on-going protein phosphorylation is required specifically for stimulation of transcription of the lipogenic genes by T3.