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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. Prohibitin genotyping of 22 breast cancer cell lines identified 17 homozygous for the B allele, 5 homozygous for the non-B allele, and no heterozygotes. Four of these cell lines were chosen for further characterization of prohibitin. In cell proliferation assays, the homozygous B breast cancer cell lines (BT-20, SK-BR-3, and MCF7) are all inhibited from traversing the cell cycle following the introduction of wild-type prohibitin transcripts. The cell line homozygous for the alternative non-B allele (BT-549) is not inhibited by transcripts. All of the breast cancer cell lines overexpress the longer form of the prohibitin mRNA (1.9 kb) and the protein. Mutational analysis of the protein-coding region detected no mutations in any of the lines. However, BT-20, SK-BR-3, and MCF7 cells are all mutated in the final 200 bases of the 3' untranslated region (3'UTR) exclusive to the 1.9-kb transcript, but BT-549 cells had no alterations in this region of the 3'UTR. Functional mapping experiments performed in the mutated SK-BR-3 line showed that the wild-type 3'UTR alone is sufficient to inhibit cell cycle progression, indicating that the antiproliferative activity of the prohibitin transcript is localized to this region. Overall, our results show that most (80%) of the cell lines derived from breast tumors have a common prohibitin genotype, suggesting that they belong to the same group of immortalized cells, group B. The results also show that the prohibitin 3'UTR exhibits the characteristics of a trans-acting regulatory RNA (riboregulator), the tumor suppressor activity of which is inactivated by mutation in group B immortalized cells.

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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. Data presented here show that the expression of a prohibitin mRNA with a long 3' untranslated region (3'UTR) and prohibitin protein is elevated in immortalized cells from all complementation groups. However, all immortalized cells classified in complementation Group B, and no cell lines in any of the other groups, are sensitive to the antiproliferative activity of wild-type prohibitin transcripts. All Group B cells are also homozygous for one of two human prohibitin alleles that are distinguishable by two distinct intron polymorphism restriction sites. Interestingly, sequence analysis of the prohibitin gene from representatives of each of the complementation groups showed that the 3'UTR from Groups A, C, and D matched wild type; however, the sequence from all four Group B cell lines differed from wild type. Functional inhibition assays on truncated wild-type mRNA transcripts as well as 3'UTR specific wild-type and mutated transcripts show that the antiproliferative activity of prohibitin resides, at least in part, in the 3'UTR. These data suggest that the prohibitin 3'UTR may function as a trans-acting regulatory RNA (riboregulator) whose tumor suppressor activity has been inactivated by mutation in Group B cells.

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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. Prohibitin is proposed to be a member of a new class of tumor suppressor genes whose inhibitory activity plays a role in the dominant senescent phenotype. Its involvement in senescence has been postulated from results obtained from such diverse systems as yeast and human diploid fibroblasts. Additional data show that prohibitin is involved in one of the limited number of pathways that results in the loss of the senescent phenotype and leads to cellular immortalization. Its involvement, however, occurs downstream in the pathway and is postulated to be part of the lost tumor suppressor activities associated with tumorigenicity.

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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. Four transcription start points (tsp) clustered within a 35-bp region were identified by rapid amplification of cDNA ends (RACE). The exon-intron boundaries in rat and human are highly conserved, with identical positioning of splice junctions. PCR analysis with conserved exon primers was used to detect length variation between rat and human PHB, and length differences were observed in all of the introns.

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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. All of the immortalized cells expressed elevated levels of prohibitin mRNA and protein. Prohibitin gene structural characterization using Southern and single-strand conformation polymorphism (SSCP) analyses distinguished two alleles. One is cleaved at a polymorphic intronic EcoRI site, exhibits an exon 6-associated SSCP, and is homozygous only in Group B cells. The other is not cleaved at the EcoRI site, has a different exon 6 SSCP pattern, and is homozygous in Groups A, C, and D. In contrast, normal cells are heterozygous for the alleles. These results suggest that prohibitin may play a role as a tumor suppressor in the immortalization of Group B cells.

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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 has antiproliferative activity and available data suggest a role in such diverse processes as normal cell cycle regulation, replicative senescence, cellular immortalization, and the development of sporadic breast tumors. Although its functional activity is presently unknown, the 30,000-Da protein has been located in the inner membrane of mitochondria, where it is postsynthetically modified, as well as on the plasma membrane of B cells, where it is associated with the IgM receptor. Prohibitin's evolutionary conservation and ubiquitous expression indicate that it is a fundamentally important gene; and current data suggest a functional role in such dissimilar processes as development, senescence, and tumor suppression.

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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. These results suggest that prohibitin is similar to the retinoblastoma gene product whose anti-proliferative activity remains active in older cells because it is not post-synthetically modified.

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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. However, several of the more than 150 peptides that were observed exhibited some cell cycle or age-related variation in radiolabeling. Ten of these were chosen for further analysis. One peptide, with an approximate molecular weight of 47 kDa and pI of 6.8 exhibited the most significant cell cycle and age-related alterations. In matrices from younger cells, incorporation into this peptide was very low in GO but increased as these cells moved through the cell cycle, with maximum incorporation occurring in S phase. As cells neared the end of their in vitro lifespan, labeling of this peptide was elevated at all stages of the cell cycle. Since many of the functional alterations observed in senescent human diploid fibroblasts are nuclear-matrix-associated activities, these results suggest that the inappropriate expression of nuclear matrix components contribute to the functional changes which characterize in vitro senescence.

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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. HiNF-D binding activity is present at high levels in rapidly proliferating cultures of human diploid fibroblasts and is reduced to less than 2% upon the cessation of proliferation induced by serum deprivation of sparsely population fibroblast cultures. Density-dependent quiescence also abolishes HiNF-D binding activity. Downregulation of transcription from the H4 gene occurs concomitant with the loss of the HiNF-D/Site II interaction, further suggesting a functional relationship between Site II occupancy and the capacity for transcription. Serum stimulation of quiescent preconfluent cells results in an increase in HiNF-D binding activity as the cells are resuming DNA synthesis and H4 histone gene transcription. Density-inhibited quiescent cells respond to serum stimulation with only a minimal increase in the HiNF-D binding activity, 30% of maximal levels. However, H4 histone gene transcription is stimulated to a level equal to that detected in extracts of the sparsely populated serum-stimulated cultures. These results suggest that there is a threshold level of HiNF-D binding activity necessary for the activation of H4 histone gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

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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. PCNA protein levels increased two- to three-fold as cells moved from G0 to S phase.

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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. Message levels were 2-3-fold lower in older populations at all stages of the cell cycle tested. PCNA protein increased from G0 through S phase in both age groups with 2-3-fold less being found in older cells. The decline in PCNA mRNA in older populations was not the result of changes in mRNA turnover or transcription. The results suggest that the reduction in PCNA expression is due to an age related alteration in a post-transcriptional regulatory function. The decline in the expression of the PCNA gene would contribute to the inability of older cells to initiate replicative DNA synthesis.

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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.1/.2 component.

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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. The decline could not be attributed to increases in glycohydrolase or the leakage of polymerase from older cell preparations. Enzyme activation with DNase I indicated that similar levels of enzyme were present in both cell lines at all in vitro ages. These results indicate that although poly(ADP-ribosyl)ation is inversely related to donor age as well as in vitro age the decrease is in response to other factors which change with increasing age.

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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. This mRNA is 2 kb in size and is expressed in eight tissues examined.

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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.2/H2A.x and H2A.z) and H3 variant synthesis (H3.1 and H3.2/H3.3) that have been used to characterize individual cell cycle states. Hydroxyurea was employed in some experiments to reduce S phase cells. The results indicate that high population doubling level (PDL) cells move through the G1 phase of the division cycle during exponential growth and exist in the G0 cell cycle state at confluence and during low serum arrest. Low PDL cells, however, exist in the G1 cell cycle state at confluence and revert to a G0 state only after maintenance as quiescent populations. This would suggest that when stimulated high PDL cells cannot enter into S phase, they revert to a GO cell cycle state.

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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. Calcium depletion did not inhibit the biosynthesis of NAD. These results suggest that mono(ADP-ribosyl)ation is involved in the regulation of cellular Ca2+ levels.

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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. These results indicate that confluent populations of older HDF are either in a different state of the cell cycle than younger cells or that they are synthesizing a different set of histone components while being maintained in the same cell cycle state at confluence. The synthesis and deposition of inappropriate histone components may alter chromatin structure in older cells and inhibit their subsequent progression through the cell cycle.

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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. The shorter repeat lengths are not due to nucleosome sliding, but result from the presence of shorter linker regions which are reduced by as much as 25% in part of the chromatin of high PDL cells.

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