Suppression of the novel growth inhibitor p33ING1 promotes neoplastic transformation.

Using a new strategy for tumour suppressor gene isolation based on subtractive hybridization and the subsequent selection of transforming 'genetic suppressor elements', we have cloned a novel gene called ING1 encoding a 33-kD protein (p33ING1) that displays characteristics of a tumour suppressor. Acute expression of transfected constructs encoding this gene inhibited cell growth while chronic expression of ING1 antisense constructs promoted cell transformation. Limited analyses of tumour cell lines show that mutation of the ING1 gene occurs in neuroblastoma cells and reduced expression was seen in some breast cancer cell lines. These results demonstrate that ING1 can act as a potent growth regulator in normal and in established cells and provide evidence for a role as a candidate tumour suppressor gene whose inactivation may contribute to the development of cancers.

Extension of the replicative life span of human diploid fibroblasts by inhibition of the p33ING1 candidate tumor suppressor.

Previous studies suggest that tumor suppressors may play significant roles in blocking the growth of cells during cellular senescence. We therefore studied the potential involvement of a novel growth inhibitor and candidate tumor suppressor gene called ING1, which we have cloned recently (I. Garkavtsev, A. Kazarov, A. Gudkov, and K. Riabowol, Nat. Genet. 14:415-420, 1996), in the process of cellular senescence. Our results show that the RNA and protein levels of ING1 were 8- to 10-fold higher in senescent cells than in young, proliferation-competent human diploid fibroblasts. Expression of the nuclear p33ING1 protein was regulated during the cell cycle, reaching maximal levels during DNA synthesis. Chronic expression of antisense ING1 RNA reproducibly resulted in extension of the proliferative life span of normal human fibroblasts by approximately seven population doublings.

The COG database: new developments in phylogenetic classification of proteins from complete genomes.

The database of Clusters of Orthologous Groups of proteins (COGs), which represents an attempt on a phylogenetic classification of the proteins encoded in complete genomes, currently consists of 2791 COGs including 45 350 proteins from 30 genomes of bacteria, archaea and the yeast Saccharomyces cerevisiae (http://www.ncbi.nlm.nih. gov/COG). In addition, a supplement to the COGs is available, in which proteins encoded in the genomes of two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and shared with bacteria and/or archaea were included. The new features added to the COG database include information pages with structural and functional details on each COG and literature references, improvements of the COGNITOR program that is used to fit new proteins into the COGs, and classification of genomes and COGs constructed by using principal component analysis.

A novel candidate tumor suppressor, ING1, is involved in the regulation of apoptosis.

We have recently cloned a novel growth inhibitor and candidate tumor suppressor called p33ING1 (I. Garkavtsev et al., Nature Genet., 14: 415-420, 1996). Because some tumor suppressors participate in the regulation of apoptosis, we hypothesized that the ING1 gene may also play a role in this process. Our results show that p33ING1 levels increase upon the induction of apoptosis in P19 teratocarcinoma cells by serum deprivation. Elevated expression of ING1 in P19 and rodent fibroblast cells containing a tetracycline-controlled human c-myc gene enhanced the extent of serum starvation-induced apoptosis. This suggests that the pathway by which ING1 modulates cell death is synergistic with Myc-dependent apoptosis. Conversely, constitutive expression of an antisense construct of INGI conferred protection against apoptosis in these cells. These data support the idea that loss of proper ING1 function may facilitate tumorigenesis, in part, by reducing the cell's sensitivity to apoptosis.

The Bloom syndrome protein interacts and cooperates with p53 in regulation of transcription and cell growth control.

Bloom syndrome is an autosomal recessive disorder associated with mutations in BLM gene encoding protein that belongs to the family of DNA helicases. It is characterized by predisposition to cancer, immunodeficiency, high sensitivity to UV and genomic instability of somatic cells. Here we show physical and functional cooperation between BLM and p53 proteins. Ectopic expression of BLM causes anti-proliferative effect in p53 wild type, but not in p53-deficient cells; p53-mediated transactivation is attenuated in primary fibroblasts from Bloom syndrome patients. BLM and p53 proteins physically interact in the cells as demonstrated in yeast and mammalian two-hybrid systems; interaction sites are mapped to 237-272 aa residues of BML and 285-340 aa of p53. Ectopic expression of the fragment of wild type BML containing p53-interactive domain suppresses p53-mediated transcription and interferes with p53-mediated growth inhibition. These observations indicate that BLM might be an important component of p53 function and suggest that Bloom Syndrome phenotype may in part be the result of the deregulation of the p53 tumor suppressor pathway.

Suppression of ING1 expression in sporadic breast cancer.

Down regulation of the ING1 candidate tumour suppressor promotes growth in soft agar and focus formation in vitro and tumour formation in vivo. ING1 encodes a nuclear, cell cycle-regulated protein, overexpression of which efficiently blocks cell growth and is capable of inducing apoptosis in different experimental systems. Here we present the first report of ING1 mutation and expression analysis in a total of 452 cancer samples. One germline missense alteration and three germline silent alterations were detected in 377 primary breast cancers while marked (2 - 10-fold) decreases in ING1 mRNA expression were seen in 44% of primary breast cancers and in ten of ten breast cancer cell lines examined. Furthermore, the majority of breast cancers (58%) showing decreased ING1 expression had metastasized to regional lymph nodes whereas only 9% of cancers with elevated ING1 expression, compared to adjacent normal tissues, were metastatic. Thus, ING1 mutation is very rare in breast or ovarian cancers, however, repression of ING1 expression frequently accompanies tumour development of breast cancer.

Molecular aspects of the relationship between cancer and aging: tumor suppressor activity during cellular senescence.

Normal cells cultured in vitro lose their proliferative potential after a finite number of doublings in a process termed replicative cellular senescence (Hayflick, 1965). The roles that growth inhibitory tumor suppressors play in the establishment and maintainence of cellular senescence have been reported in many different systems. The Rb and p53 tumor suppressors are examples of growth inhibitors that lose the ability to be regulated and are constantly activated during senescence. Other proteins that inhibit the initiation of DNA synthesis in early passage fibroblasts and that link the action of tumor suppressors with the cell cycle machinery, are also expressed at higher levels in senescent cells. For example, the increased expression of the cyclin-dependent kinase inhibitor p16 may contribute to arresting the growth of senescent cells. Identification and characterization of additional genes encoding growth inhibitors that are upregulated in senescent cells, such as the recently isolated p33ING1 protein, should provide a better understanding of the "aging program" that ceases to operate in the generation of immortal cancer cells.

Specific monoclonal antibody raised against the p33ING1 tumor suppressor.

An IgG1 mouse monoclonal antibody (CAb1) was produced against human recombinant p33ING1. The antibody is able to recognize native and denatured antigen in ELISA and Western blot protocols, respectively. CAb1 can be used to specifically detect the p33ING1 protein in dot blot and Western immunoblot protocols of both human and mouse cell lysates. In addition, this antibody is also useful for cellular localization of native and ectopically overexpressed p33ING1 protein by indirect immunofluorescence.

Integrated map of the Schizosaccharomyces pombe genome.

A restriction map of the entire Schizosaccharomyces pombe genome was constructed using two restriction enzymes (BamHI and PstI) that recognize 6 bp. The restriction map contains 420 minimally overlapping clones (miniset) and has 22 gaps. We located 126 genes, marker fragments of DNA (NotI and SfiI linking clones), and 36 transposable elements by hybridization to unique restriction fragments.

Cellular localization and chromosome mapping of a novel candidate tumor suppressor gene (ING1).

A novel gene called ING1 encoding a 33-kDa protein that is an inhibitor of cell growth and a candidate tumor suppressor has been recently isolated (Garkavtsev et al., 1996). Here we show, using indirect immunofluorescence, that the protein (p33ING1) is located in the nucleus, which is consistent with its proposed role as a growth regulator. In addition, we show that a genomic probe to human ING1 localizes to chromosome 13 at q33-->q34 by fluorescence in situ hybridization. This candidate tumor suppressor gene is located near a chromosome region which has been reported to be a site for translocation and deletion in gastric cancers and head and neck squamous carcinomas.

Variability of human rRNA genes: inheritance and nonrandom chromosomal distribution of structural variants of nontranscribed spacer sequences.

Human rRNA genes contain variable regions, one of which is located in nontranscribed spacers (NTSs) closely downstream from the 3'-end of the transcribed region. This polymorphism may be detected by means of blot hybridization analysis as a set of distinct restriction fragments corresponding to this part of the rRNA genes. We have analyzed DNA of 51 individuals and found eight structural NTS variants of this region; two of these were common to all individuals analyzed, and six others were found in different combinations and with different frequencies. The copy number of each variant also differed but was not less than 10-20 copies per cell. The analysis of DNA isolated from leukocytes of the members of 11 families indicated that some of the structural variants (of the NTS region) are inherited as a single Mendelian locus. We propose that rRNA genes that belong to one particular structural variant form clusters on separate chromosomes. To test this proposition, we developed a combined method, including AgNO3-staining of chromosomes, in situ hybridization, and DNA analysis with methylation-sensitive restrictases, and used it for study of persons who had methylated rRNA genes located on AgNO3-negative nucleolar organizers. It was found that in three of four cases methylated genes really belonged to one structural variant. This approach may be used for detailed localization of separate classes of NTS structural variants of human rRNA genes.

Increased activity of p53 in senescing fibroblasts.

The p53 tumor-suppressor protein binds DNA and activates the expression of a 21-kDa protein that inhibits both the activity of cyclin-dependent kinases and the function of proliferating cell nuclear antigen. Since p21 expression has been reported to increase 10- to 20-fold as human diploid fibroblasts lose the ability to replicate, we examined the expression and activity of p53 during replicative aging. Similar levels of total p53 mRNA and protein were expressed in low-passage (young) and high-passage (old) cells but both DNA binding activity in vitro and transcriptional activity of p53 in vivo were increased severalfold in high-passage cells. While the basis of increased p53 activity is presently unclear, it is not correlated with differential phosphorylation or changes in p53-mouse double minute 2 gene product interactions. These results provide evidence for the activation of a protein involved in the control of cell cycle checkpoints during cellular aging, in the absence of increased expression.

The candidate tumour suppressor p33ING1 cooperates with p53 in cell growth control.

The candidate tumour-suppressor gene ING1 has been identified by using the genetic suppressor element (GSE) methodology. ING1 encodes a nuclear protein, p33ING1, overexpression of which inhibits growth of different cell lines. The properties of p33ING1 suggest its involvement in the negative regulation of cell proliferation and in the control of cellular ageing, anchorage dependence and apoptosis. These cellular functions depend largely on the activity of p53, a tumour-suppressor gene that determines the cellular response to various types of stress. Here we report that the biological effects of ING1 and p53 are interrelated and require the activity of both genes: neither of the two genes can, on its own, cause growth inhibition when the other one is suppressed. Furthermore, activation of transcription from the p21/WAF1 promoter, a key mechanism of p53-mediated growth control, depends on the expression of ING1. A physical association between p33ING1 and p53 proteins has been detected by immunoprecipitation. These results indicate that p33ING1 is a component of the p53 signalling pathway that cooperates with p53 in the negative regulation of cell proliferation by modulating p53-dependent transcriptional activation.

Structure and regulation of the mouse ing1 gene. Three alternative transcripts encode two phd finger proteins that have opposite effects on p53 function.

The human ING1 gene encodes nuclear protein p33(ING1), previously shown to cooperate with p53 in cell growth control (Garkavtsev, I., Grigorian, I. A., Ossovskaya, V. S., Chernov, M. V., Chumakov, P. M., and Gudkov, A. V. (1998) Nature 391, 295-298). p33(ING1) belongs to a small family of proteins from human, mouse, and yeast of approximately the same size that show significant similarity to one another within the C-terminal PHD finger domain and also contain an additional N-terminal region with subtle but reliably detectable sequence conservation. Mouse ing1 is transcribed from three differently regulated promoters localized within a 4-kilobase pair region of genomic DNA. The resulting transcripts share a long common region encoded by a common exon and differ in their 5'-exon sequences. Two transcripts are translated into the same protein of 185 amino acids, the mouse equivalent of the human p33(ING1), while the third transcript encodes a longer protein that has 94 additional N-terminal amino acids. Overexpression of the longer protein interferes with the accumulation of p53 protein and activation of p53-responsive promoters after DNA damage. Between the two products of ing1, only the longer one forms a complex with p53 detectable by immunoprecipitation. These results indicate that a single gene, ing1, encodes both p53-suppressing and p53-activating proteins that are regulated by alternative promoters.

A 13 kb resolution cosmid map of the 14 Mb fission yeast genome by nonrandom sequence-tagged site mapping.

We present the application of a nonrandom sequence-tagged site (STS) content detection method in mapping an entire genome, that of fission yeast. The novelty of our strategy is in the use of STS probes made from both ends of cosmid clones, selected on the basis of "sample without replacement" (only library clones that show no previous positive hybridization are selected and made into probes). We developed powerful techniques, based on consistency analysis, for error detection and contig assembly. In addition, we probed our library with genetically mapped markers and Notl or Sfil linking clones, thereby anchoring contigs onto chromosomes. Our map contains more than 1000 sites, including genes (most were previously unmapped), occurrences of known repetitive elements, and Notl-Sfil restriction sites.