Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression.

BACKGROUND: The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers. METHODS: We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures. We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. All statistical tests were two-sided. RESULTS: RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 64% of breast cancer lines, in 30% of primary NSCLCs, and in 49% of primary breast tumors but in none of the nonmalignant lung tissues. RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P =.046). Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity. CONCLUSION: RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.

HRR25, a putative protein kinase from budding yeast: association with repair of damaged DNA.

In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH2-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.

A brief consideration of the SOS inducing signal.

SOS functions are induced in E. coli by treatments that damage cellular DNA or interrupt its synthesis. The biochemical basis of induction is activation of the specific proteolytic activity of recA protein, which then inactivates the lexA repressor. We discuss the development of the inducing signal in the cell.

Catabolism of L-tyrosine in Trichosporon cutaneum.

Protocatechuic acid was a catabolite in the degradation of L-tyrosine by Trichosporon cutaneum. Intact cells oxidized to completion various compounds proposed as intermediates in this conversion, but they did not readily oxidize catabolites of the homogentisate and homoprotocatechuate metabolic pathways, which are known to function in other organisms. Cell extracts converted tyrosine first to 4-hydroxycinnamic acid and then to 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid. The proposed hydration product of 4-hydroxycinnamic acid, namely, beta-(4-hydroxyphenyl)-hydracrylic acid, was synthesized chemically, and its enzymatic degradation to 4-hydroxybenzaldehyde was shown to be dependent upon additions of adenosine triphosphate and coenzyme A. The hydroxylase that attacked 4-hydroxybenzoate showed a specific requirement for reduced nicotinamide adenine dinucleotide phosphate. Protocatechuate, the product of this reaction, was oxidized by cell extracts supplemented with reduced nicotinamide adenine dinucleotide or, less effectively, with reduced nicotinamide adenine dinucleotide phosphate, but these extracts contained no ring fission dioxygenase for protocatechuate. Evidence is presented that the principal hydroxylation product of protocatechuate was hydroxyquinol, the benzene nucleus of which was cleaved oxidatively to give maleylacetic acid.