Richard Sobel

Newly synthesized histone H4 is deposited in a diacetylated isoform in a wide variety of organisms. In Tetrahymena a specific pair of residues, lysines 4 and 11, have been shown to undergo this modification in vivo. In this report, we demonstrate that the analogous residues, lysines 5 and 12, are acetylated in Drosophila and HeLa H4. These data strongly suggest that deposition-related acetylation sites in H4 have been highly, perhaps absolutely, conserved. In Tetrahymena and Drosophila newly synthesized histone H3 is also deposited in several modified forms. Using pulse-labeled H3 we have determined that, like H4, a specific, but distinct, subset of lysines is acetylated in these organisms. In Tetrahymena, lysines 9 and 14 are highly preferred sites of acetylation in new H3 while in Drosophila, lysines 14 and 23 are strongly preferred. No evidence has been obtained for acetylation of newly synthesized H3 in HeLa cells. Thus, although the pattern and sites of deposition-related acetylation appear to be highly conserved in H4, the same does not appear to be the case for histone H3.

Heterochromatin in metazoans induces transcriptional silencing, as exemplified by position effect variegation in Drosophila melanogaster and X-chromosome inactivation in mammals. Heterochromatic DNA is packaged in nucleosomes that are distinct in their acetylation pattern from those present in euchromatin, although the role these differences play in the structure of heterochromatin or in the effects of heterochromatin on transcriptional activity is unclear. Here we report that, as observed in the facultative heterochromatin of the inactive X chromosome in female mammalian cells, histones H3 and H4 in chromatin spanning the transcriptionally silenced mating-type cassettes of the yeast Saccharomyces cerevisiae are hypoacetylated relative to histones H3 and H4 of transcriptionally active regions of the genome. By immunoprecipitation of chromatin fragments with antibodies specific for H4 acetylated at particular lysine residues, we found that only three of the four lysine residues in the amino-terminal domain of histone H4 spanning the silent cassettes are hypoacetylated. Lysine 12 shows significant acetylation levels. This is identical to the pattern of histone H4 acetylation observed in centric heterochromatin of D. melanogaster. These two observations provide additional evidence that the silent cassettes are encompassed in the yeast equivalent of metazoan heterochromatin. Further, mutational analysis of the amino-terminal domain of histone H4 in S. cerevisiae demonstrated that this observed pattern of histone H4 acetylation is required for transcriptional silencing. This result, in conjunction with prior mutational analyses of yeast histones H3 and H4, indicates that the particular pattern of nucleosome acetylation found in heterochromatin is required for its effects on transcription and is not simply a side effect of heterochromatin formation.

PURPOSE: This is part 1 of a 2-part review. Research into the molecular mechanisms underlying the various aspects of prostate cancer (PCa) requires the use of in vivo and in vitro model systems. In the last few years many new cell lines have been established by investigators from primary tissue sources and clonal derivatives of previously established lines. Therefore, the purpose of this 2-part review is to catalogue the current human cell lines developed for PCa research, as reported in the literature. Part 1 includes tissue culture cell lines derived from metastases, primary tumors and nonadenocarcinomas that were established without the use of transgenes. It also includes a section describing lines that have been contaminated with other lines, shown not to be of prostatic origin or whose identity is being challenged. MATERIALS AND METHODS: Prostate cell lines included in this review were identified by extensive searching of the literature using several strategies, including PubMed searches and book chapter reviews. RESULTS: In total we describe the derivation, phenotype, genotype and characterization of molecular markers expressed by approximately 200 lines and sublines used in PCa research, including those derived from primary tumors, metastases and normal prostate tissue. We paid particular attention to the expression of prostate specific antigen, androgen receptor, cytokeratins and other molecular markers used to indicate the status of PCa and the prostatic lineage of a given line. In an attempt to provide PCa researchers with a resource of information regarding new and established cell lines we have also created an online database of these PCa cell lines freely accessible via the World Wide Web at http://www.CaPCellLines.com. The web based interface allows researchers to peruse and print information regarding cell lines, add new cell lines and update or add new information regarding established cell lines. CONCLUSIONS: This compendium of cell lines currently used in PCa research combined with access to our on-line database provides researchers with a continually updated and valuable resource for investigating the molecular mechanisms of PCa.

PURPOSE: This is part 2 of a 2-part review. Research into the molecular mechanisms underlying the various aspects of prostate cancer (PCa) requires the use of in vivo and in vitro model systems. In the last few years many new cell lines have been established by investigators from primary tissue sources and clonal derivatives of previously established lines. Therefore, the purpose of this 2-part review is to catalogue the current human cell lines developed for PCa research, as reported in the literature. Part 2 describes tissue culture cell lines derived by the insertion of transgenes, including human telomerase reverse transcriptase, SV40 T antigen and human papillomavirus genes. Part 2 also includes xenograft lines that require propagation and passage in vivo in mice. MATERIALS AND METHODS: Prostate cell lines included in this review were identified by extensive searching of the literature using several strategies, including PubMed searches and book chapter reviews. RESULTS: In total we describe the derivation, phenotype, genotype and characterization of molecular markers expressed by approximately 200 lines and sublines used in PCa research, including ones derived from primary tumors, metastases and normal prostate tissue. We paid particular attention to the expression of prostate specific antigen, androgen receptor, cytokeratins and other molecular markers used to indicate the status of PCa and the prostatic lineage of a given line. In an attempt to provide PCa researchers with a resource of information regarding new and established cell lines we have also created an online database of these PCa cell lines freely accessible via the World Wide Web at http://www.CaPCellLines.com. The web based interface allows researchers to peruse and print information regarding cell lines, add new cell lines and update or add new information regarding established cell lines. CONCLUSIONS: This compendium of cell lines currently used in PCa research combined with access to our on-line database provides researchers with a continually updated and valuable resource for investigating the molecular mechanisms of PCa.

During periods of active DNA replication and chromatin assembly, newly synthesized histone H4 is deposited in a diacetylated form. In Tetrahymena, a specific pair of residues, lysines 4 and 11, has been shown to undergo this modification in vivo (Chicoine, L. G., Schulman, I. G., Richman, R., Cook, R. G., and Allis, C. D. (1986) J. Biol. Chem. 261, 1071-1076). Presumably, this reaction is catalyzed, at least in part, by histone acetyltransferases (HAT) of the B type, cytoplasmic enzymes displaying strong preference for free, non-chromatin-bound H4. To investigate which lysines are preferred acetylation sites in H4 from other organisms, a cytoplasmic HAT B activity was prepared from Drosophila embryos and used to acetylate H4 from several species. When H4 or synthetic, NH2-terminal peptides from Tetrahymena were used as unblocked substrates, direct microsequence analyses showed that [3H]acetate was preferentially incorporated at lysine 11 with little, if any, incorporation at other conserved, acetylatable lysines. Drosophila H4 was chemically deblocked following its acetylation in vitro using conditions that do not deacetylate internal lysines. Direct sequence analysis verified the correct NH2-terminal sequence of Drosophila H4 and demonstrated that [3H]acetate incorporation occurred preferentially on lysine 12, the residue analogous to lysine 11 in Tetrahymena. These data show remarkable preference for lysine 11/12 by the Drosophila HAT B activity in vitro and provide support for the assertion that this activity functions to acetylate new H4, at least in part, for deposition and chromatin assembly in vivo. Since most H4s, like Drosophila, are blocked at their amino termini by an acetylthreonine or acetylserine, our results demonstrate that this deblocking and microsequencing strategy can be used to study acetylation site utilization in H4 and presumably other core histones NH2 terminally blocked with these residues.