Prohibitin in breast cancer cell lines: loss of antiproliferative activity is linked to 3' untranslated region mutations.

The evolutionarily conserved prohibitin gene is located on human chromosome 17q21, and two alleles have been identified. Our previous studies characterizing prohibitin in immortalized cells, classified into four complementation groups (A-D) based on the ability of whole-cell hybrids to become senescent, have suggested that it has tumor suppressor activity in group B cells. Only the cell lines assigned to group B are sensitive to the antiproliferative activity of prohibitin, and all are homozygous for an allele designated B because of its exclusive association with this group.

The 3' untranslated region of prohibitin and cellular immortalization.

We have been studying the role of the evolutionarily conserved prohibitin gene in cellular immortalization and tumor suppression. Immortalized human cells are classified into four complementation groups (A, B, C, and D) based on the ability of fusion hybrids to become senescent. The present study expands our preliminary evidence showing that the antiproliferative activity of prohibitin is only effective in immortalized Group B cells and normal cells.

Prohibitin and the senescent phenotype.

Prohibitin is an evolutionarily conserved gene that has antiproliferative activity, is ubiquitously expressed, and appears to be essential for cell survival. The gene codes for a 30 kD, post-synthetically modified protein located primarily in the mitochondria. It functionally inhibits cell cycle traverse and DNA synthesis, but its mechanism of action is presently unknown.

Regions of evolutionary conservation between the rat and human prohibitin-encoding genes.

We have analyzed and compared the 5' promoter region, the intron structure and the exon-intron flanking sequences in the rat and human prohibitin-encoding genes (PHB). Comparative analysis of a 350-nt region immediately 5' to and including the first exon identifies eight highly conserved regions, four of which correspond to binding sites for known transcriptional control proteins (CCAAT box, 'SV40' site and two Sp1 sites). The promoter lacks a TATA box.

Prohibitin antiproliferative activity and lack of heterozygosity in immortalized cell lines.

Experiments were performed to determine whether prohibitin, an evolutionarily conserved gene with antiproliferative activity, has a role in cellular immortalization. A cell proliferation assay was used to examine one human cell line from each of four established immortal complementation groups, termed A, B, C, and D, and a normal human diploid fibroblast line. Only normal and Group B cells were inhibited from traversing the cell cycle after introduction of wild-type prohibitin transcript.

Prohibitin: potential role in senescence, development, and tumor suppression.

Prohibitin is an evolutionarily conserved gene with homologues found in organisms ranging from yeast to man. In man the gene is located on chromosome 17 at q21. The deduced amino acid sequences of the protein products from mouse and rat are identical; and these differ from the human protein sequence by a single conserved amino acid.

Prohibitin expression during cellular senescence of human diploid fibroblasts.

Prohibitin is an evolutionarily conserved gene postulated to possess tumor suppressor activity and to contribute to the limited lifespan of human diploid fibroblast-like cells. Prohibitin mRNA and protein expression and its ability to become post-translationally modified were determined in human diploid fibroblast-like cells of different in vitro ages. The expression of prohibitin mRNA and protein changes little with increasing in vitro age; however, its protein product is post-synthetically modified in younger but not older cells.

Nuclear matrix composition and in vitro cellular senescence.

The synthesis of nuclear matrix components from human diploid fibroblasts of different in vitro ages was analyzed. Radiolabeled nuclear matrices were prepared from human diploid fibroblasts at various stages of the cell cycle, and their components were separated by two dimensional electrophoresis. The same general electrophoretic pattern was observed at all cell cycle points analyzed, regardless of in vitro age.

Multiple mechanisms regulate the proliferation-specific histone gene transcription factor HiNF-D in normal human diploid fibroblasts.

The proliferation-specific transcription factor complex HiNF-D interacts with sequence specificity in a proximal promoter element of the human H4 histone gene FO108, designated Site II. The occupancy of Site II by HiNF-D has been implicated in proper transcription initiation and as a component of the cell cycle regulation of this gene. In the present study we have investigated the role of the HiNF-D/Site II interaction in controlling the level of H4 histone gene transcription during modifications of normal cellular growth.

Expression of proliferating cell nuclear antigen during the cell cycle of human diploid fibroblasts.

Proliferating cell nuclear antigen mRNA levels were determined in human diploid fibroblasts as they progressed through the cell cycle. PCNA message levels were low at G0, gradually increased following entrance into G1, peaked at G1/S, and declined during S phase. PCNA mRNA was determined to have a half life of 12 hours when cells were blocked at the G1/S interface.

Age related decline in the expression of proliferating cell nuclear antigen in human diploid fibroblasts.

Proliferating cell nuclear antigen mRNA and protein levels were determined in human diploid fibroblasts of different in vitro ages as they progressed through the cell cycle. Cells were analyzed at G0; at various stages of G1, including the G1/S interface; and during S. At all in vitro ages, PCNA message levels were low to undetectable at G0, were evident 8 to 12 h following entrance into G1, peaked at G1/S and declined during S phase.

Histone H2A variant synthesis in aging human diploid cells.

The synthesis of the various classes of core particle histones was determined in human diploid fibroblast-like cells of different in vitro ages as they were stimulated to enter the cell cycle. Histone H2A synthesis in older populations was lower during G1 but was similar to younger cells at G0/G1 and S phases. The reduced synthesis during G1 was primarily the result of a decline in the synthesis of the H2A.

Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells.

Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells.

Decline of poly(ADP-ribosyl)ation during in vitro senescence in human diploid fibroblasts.

Poly(ADP-ribose) polymerase activity was determined at various times during the in vitro life span of two human diploid fibroblast-like cell lines of different donor ages. The cell lines differed in their ability to transfer ADP-ribose, with cells from an embryonic donor exhibiting 2 to 3 times the activity found in cells obtained from a newborn donor. The activity in both cell lines decreased by 30-60% as the cells moved through their in vitro life spans.

Isolation of a cDNA that hybrid selects antiproliferative mRNA from rat liver.

Studies of chromosome loss in inherited cancers, of fusions between proliferating and quiescent cells, and of microinjection of RNA from quiescent cells into proliferation competent cells have all provided evidence for antiproliferative genes in mammalian cells. In this report, we describe a partial cDNA clone isolated on the basis of its preferential hybridization to RNA from normal versus regenerating rat liver. The corresponding mRNA, enriched by hybrid selection, was microinjected into normal human diploid fibroblasts in cell culture, resulting in a 53% decrease in the fraction of nuclei incorporating tritiated thymidine.

The effects of in vitro age and culture state on histone variant synthesis in human diploid fibroblasts.

Histone variant synthesis patterns from human diploid fibroblast-like cells of different in vitro ages were determined during exponential growth, at confluence, and during low serum arrest. The results are reported as the ratios of H2A variant synthesis (H2A.1 and H2A.

Stimulation of mono (ADP-ribosyl)ation by reduced extracellular calcium levels in human fibroblasts.

Lowering extracellular calcium in cultures of human diploid fibroblast-like cells caused a rapid depletion of NAD pools. This loss of NAD was reversed by restoring extracellular Ca2+ and was inhibited by 3-aminobenzamide, an inhibitor of ADP-ribosyl transfer reactions. The concentrations of 3-aminobenzamide needed to inhibit the loss of NAD were consistent with those required to inhibit cellular mono(ADP-ribosyl) rather than poly(ADP-ribosyl) reactions.

Histone variant synthesis patterns in confluent populations of human diploid cells of different in vitro ages.

Histone variant synthesis patterns have been used to determine the cell cycle state of cultured cells. H2A and H3 histone variant synthesis patterns from confluent populations of human diploid fibroblast-like cells of different in vitro ages were determined and compared as ratios. In the absence of significant levels of DNA synthesis, there was an age related increase in the H2A ratio while the H3 ratio remained constant.

Histone gene expression remains coupled to DNA synthesis during in vitro cellular senescence.

Despite a decrease in the extent to which confluent monolayers of late compared to early passage CF3 human diploid fibroblasts can be stimulated to proliferate, the time course of DNA synthesis onset is similar regardless of the in vitro age of the cells. A parallel and stoichiometric relationship is maintained between the rate of DNA synthesis and the cellular levels of histone mRNA independent of the age of the cell cultures. Furthermore, DNA synthesis and cellular histone mRNA levels decline in a coordinate manner after inhibition of DNA replication by hydroxyurea treatment.

Histone gene stability during cellular senescence.

The extent to which human histone gene organization is conserved during the in vitro lifespan of human diploid fibroblast-like cells was determined by comparing the restriction patterns of a human H4 and an H3 histone gene from cells of various in vitro ages. No age related change in the organization of these two genes was detected.

Changes in the higher order organization of DNA during aging of human fibroblast-like cells.

Nucleosome spacing (DNA repeat length) was determined in human diploid fibroblast-like cells (HDF) of different in vitro ages following the electrophoretic separation of micrococcal nuclease digestion products. The results indicate that a heterogeneity of DNA repeat lengths is present in HDF of all in vitro ages. In older cells the organization of part of the DNA is conserved, but a greater proportion of shorter repeats is evident.

Human histone gene organization. Identification of a histone gene polymorphism prevalent in a black population.

Analysis of the restriction enzyme digests of total genomic DNAs from a broad spectrum of human cell lines and from individuals with different genetic backgrounds, by hybridization with a series of cloned human histone sequences, indicated restriction site polymorphisms (RSPs) for two adjacent human histone genes which reside on chromosome 1. In most cell lines and individuals examined we observed a single 2.05 kb H4 histone HindIII fragment and a 7.

Variable transglutaminase activity in human diploid fibroblasts during in vitro senescence.

The specific activity of transglutaminase (TGase) was followed in human diploid fibroblasts (HDF) as a function of in vitro age. It was determined that at least 90% of the TGase activity was found in a soluble fraction at all in vitro ages; but the activity was variable with age. It was high in cells that had completed less than 50% of their lifespan (%LSC), declined to a minimum between 60 and 85% LSC, and again became elevated at more than 90% LSC.