Susan C. Evans

An alternatively spliced HDM2 product increases p53 activity by inhibiting HDM2

The human counterpart hdm2 of the murine double-minute 2 (mdm2) gene encodes a 90-kD protein (HDM2) that inhibits the function of the p53 tumor suppressor. Hdm2 is amplified in approximately 30% of sarcomas, leading to overproduction of HDM2 and inactivation of p53. Using immunohistochemistry to screen a panel of human tumors for HDM2 overproduction, we detected high levels of HDM2 in the cytoplasm in 25% of lung tumors as opposed to its normal localization in the nucleus. These samples contained full-length hdm2 and several alternate-splice forms of hdm2 mRNA. Sequence analysis revealed deletions in the alternate-splice forms of the p53 binding domain and absence of a nuclear localization signal. In transient transfection assays, one of the alternate-splice forms, HDM2ALT1, bound and sequestered full-length HDM2 in the cytoplasm. In addition, the binding of HDM2ALT1 to HDM2 inhibited the interaction of HDM2 with p53, thus enhancing p53 transcriptional activity. These data suggest the existence of another level of regulation of HDM2 which increases the activity of p53.

The Li-Fraumeni syndrome: An inherited susceptibility to cancer

The Li-Fraumeni syndrome is a rare autosomal-dominant disease whose hallmark is a predisposition to a wide range of cancers among members of a family. Many of these families have a germline mutation within the tumor suppressor gene TP53, which encodes the p53 protein. The inheritance of a mutant TP53 allele results in a 25-fold increase in the chance of developing cancer by 50 years of age, compared with the general population. TP53 mutations are also very common in the development of somatic tumors. This article reviews the biological and biochemical role of p53 in the susceptibility to cancer in Li-Fraumeni syndrome.

CELL SURFACE GALACTOSYLTRANSFERASE : CURRENT ISSUES

The diversity of carbohydrate structures both amazes and overwhelms us, as does the diversity of the enzymes that are responsible for their synthesis. Cells express only a limited subset of the glycosyltransferases and glycosidases that are available to them, and do so in tissue-specific and spatiallyspecific patterns. In this regard, how particular enzymatic components of the glycosylation apparatus become and remain segregated in specific subcellular organelles is a question that impacts on issues ranging from cell biology to pharmaceutical development. Clearly, amino acid sequences contained within and adjacent to the transmembrane domain are responsible, at least in part, for the selective retention of specific glycosyltransferases to the Golgi compartments. The specific amino acid residues that are required, and how they cooperate with cytosolic proteins to localize unique glycosyltransferases to defined membrane organelles continues to be an area of intense study [1—3]. Compounding this complexity of glycosyltransferase expression, are studies that show that some glycosyltransferases are localized on the plasma membrane in addition to their conventional Golgi location [4, 5]. This prompted the hypothesis that glycosyltransferases may function as cell adhesion molecules by binding their complementary

Exclusion of a p53 germline mutation in a classic Li-Fraumeni syndrome family

Abstract Li-Fraumeni syndrome (LFS) is characterized by a high risk of sarcomas, early onset of breast cancer, and a diversity of other cancers occurring as multiple primary tumors in multiple family members. In many families with LFS, germline mutations within the tumor-suppressor gene p53 have been identified. However, mutations in p53 have not been detected in approximately 30% of LFS families. To address the possibility either that p53 mutations were being missed or that another predisposing gene is altered in LFS, we used a variety of methods to accurately determine the p53 status in a large LFS kindred. A transcriptional activation assay on exons 4–10 of p53 excluded a mutation within the DNA-binding domain of p53. Single-stranded conformational-polymorphism analysis, using intronic primers and sequencing of all the coding exons and intron/exon junctions, also yielded no mutations. Finally, linkage analysis excluded potential mutations in the noncoding regions of p53. Our findings exclude the presence of a p53 germline mutation in a classic LFS family.

Mutational analysis of the cytoplasmic domain of β1,4- galactosyltransferase I: influence of phosphorylation on cell surface expression

β1,4-Galactosyltransferase I (GalT I) exists in two subcellular compartments where it performs two distinct functions. The majority of GalT I is localized in the Golgi complex where it participates in glycoprotein biosynthesis; however, a small portion of GalT I is expressed on the cell surface where it functions as a matrix receptor by binding terminal N-acetylglucosamine residues on extracellular glycoside ligands. The GalT I polypeptide occurs in two alternate forms that differ only in the length of their cytoplasmic domains. It is thought that the longer cytoplasmic domain is responsible for GalT I function as a cell surface receptor because of its ability to associate with the detergent-insoluble cytoskeleton. In this study, we demonstrate that the long GalT I cytoplasmic and transmembrane domains are capable of targeting a reporter protein to the plasma membrane, whereas the short cytoplasmic and transmembrane domains do not have this property. The surface-localized GalT I reporter protein partitions with the detergent-insoluble pool, a portion of which co-fractionates with caveolin-containing lipid rafts. Site-directed mutagenesis of the cytoplasmic domain identified a requirement for serine and threonine residues for cell surface expression and function. Replacing either the serine or threonine with aspartic acid reduces surface expression and function, whereas substitution with neutral alanine has no effect on surface expression or function. These results suggest that phosphorylation negatively regulates GalT I function as a surface receptor. Consistent with this, phosphorylation of the endogenous, full-length GalT I inhibits its stable expression on the cell surface. Thus, the 13 amino acid extension unique to the long GalT I isoform is required for GalT I expression on the cell surface, the function of which is regulated by phosphorylation.

Regulation of hdm2 by Stress-Induced hdm2alt1 in Tumor and Nontumorigenic Cell Lines Correlating with p53 Stability

Alternative and aberrant splicing of hdm2 occurs in tumor and normal tissues. However, the factors that induce these splice variants and whether they are translated to protein products in vivo is unknown, making it difficult to decipher which of these hdm2 transcripts have a normal physiologic function or contribute to carcinogenesis. We investigated the conditions that induce this post-transcriptional modification of hdm2 in tumor and nontumorigenic cell lines. We showed that UV and gamma radiation as well as cisplatin treatment induced alternative splicing of hdm2, which resulted in a single splice variant, hdm2(alt1), irrespective of the cell type. Interestingly, the mechanism of UV-induced splicing is independent of p53 status. Immunoanalysis revealed that, after UV radiation, HDM2(ALT1) protein was expressed and interacted with HDM2 that correlated to increased p53 protein levels and its accumulation in the nucleus, whereas HDM2 localized more to the cytoplasm with a decrease in its RNA and protein level. We propose that stress-induced HDM2(ALT1) regulates HDM2 at two levels, RNA and protein, further modulating the p53-HDM2 interaction or interactions of HDM2 with other cell cycle regulatory proteins. This kind of regulation may possibly restrict oncogenic functions of HDM2 and contribute to the many protective responses triggered by certain stress signals. Our data imply that HDM2(ALT1) possesses a normal physiologic function in damaged cells, perhaps facilitating cellular defense.

Isolation of the Aspergillus nidulans sudD gene and its human homologue

We have been studying the heat-sensitive bimD6 mutation of Aspergillus nidulans. At a restrictive temperature, the chromosomes of bimD6 mutant strains fail to attach properly to the spindle microtubules, and the mutant also displays a high rate of chromosome loss. We previously cloned the sudA gene, an extragenic suppressor of the heat-sensitive bimD6 mutation and showed that it coded for a DA-box or SMC protein. SMC proteins have been demonstrated to function in chromosome condensation, segregation and global gene regulation. We have now cloned the sudD gene, another of the extragenic suppressor genes of the bimD6 mutation. The predicted SUDD protein is the founding member of a widely expressed protein family. Similar proteins are found in sequence databases for Saccharomyces cerevisiae, Caenorhabditis elegans, mammals and four species of archaebacteria. We have also cloned and sequenced a human cDNA that encodes the human homologue of SUDD and mapped the gene to 18q11.2. The predicted SUDD proteins from A. nidulans, Homo sapiens and S. cerevisiae all share a variety of features. The predicted proteins are approximately 60000Da in mass and have a serine-plus-threonine content of about 11%. The evolutionary conservation of the proteins suggests an ancient origin and conserved function for these proteins.

Insulin receptor signaling activated by penta-O-galloyl-α-d-glucopyranose induces p53 and apoptosis in cancer cells

Abstractp53 is essential for cell cycle arrest and apoptosis induction while insulin receptor (IR) signaling is important for cell metabolism and proliferation and found upregulated in cancers. While IR has recently been found to be involved in apoptosis, p53 induction or apoptosis mediated through IR signaling activation has never been documented. Here, we report that the IR signaling pathway, particularly the IR-MEK pathway, mediates biological and biochemical changes in p53 and apoptosis in tumor cells. Specifically, natural compound penta-O-galloyl-α-d-glucopyranose (α-PGG), a previously characterized IR signaling activator, induced apoptosis in RKO cells without significantly affecting its normal counterpart FHC cells. α-PGG induced apoptosis in RKO cells through p53, Bax and caspase 3. Importantly, α-PGG’s ability to elevate p53 was diminished by IR inhibitor and IR-siRNA, suggesting a non-conventional role of IR as being involved in p53 induction. Further studies revealed that α-PGG activated MEK, a downstream signaling factor of IR. Blocking MEK significantly suppressed α-PGG-induced p53 and Bax elevation. All these results suggested that α-PGG induced p53, Bax, and apoptosis through the IR-MEK signaling pathway. The unique activity of α-PGG, a novel IR phosphorylation and apoptosis inducer, may offer a new therapeutic strategy for eliciting apoptotic signal and inhibiting cancer growth.

A novel genetic modifier of p53, mop1, results in embryonic lethality.

The heterogeneity that occurs in the tumor spectrum and latency in Li-Fraumeni syndrome (LFS) patients with inherited mutations in p53 suggest risk modifiers at loci other than the major gene. We developed a mouse model to investigate these risk modifiers. Inbred CE/J mice, which succumb to multiple types of tumors similar to those found in LFS, were crossed with the p53-null 129/Sv (129-Trp53tm1Tyj) mouse. In this cross, we uncovered evidence for a genetic modifier of p53, mop1, based on an unexpected mix of genotypes in the F2 progeny from Mendelian expectations. A model in which a recessive CE/J allele in combination with p53 heterozygosity or homozygosity results in lethality most closely fits the data. Using simple-sequence length polymorphism analysis of the entire genome, we identified a putative chromosomal region for this modifier of p53 on mouse chromosome 11 centromeric to p53.

Ovca1, a candidate gene of the genetic modifier of Tp53, Mop2, affects mouse embryonic lethality.

In this study, we show genetic modifier genes of Tp53 that can exacerbate embryonic abnormalities. Using a mouse model in which CE/J mice were crossed with the Tp53‐null 129/Sv (129‐Trp53tm1 Tyj) mice, a subset of Tp53+/− and −/− male and female embryos died during gestation. Our hypothesis, based on the genotypes of survivors, is that two genetic modifiers and a Tp53 null allele lead to an increase in embryonic lethality. We previously identified a recessive modifier (Mop1) from CE/J mice on chromosome 11 centromeric to Tp53. We have uncovered a dominant modifier (Mop2) from 129/Sv mice telomeric to Tp53. We discovered a polymorphic change (321P→321S) of Ovca1 within the Mop2 locus of CE/J mice. This polymorphism increased both mRNA and protein levels of OVCA1 in various tissues. CE/J primary cells cultured from different tissues proliferated more rapidly than 129/Sv cells. In addition, CE/J cells cycled while 129/Sv cells had a higher arrest in the G1 phase. Transfection of Ovca1 containing the 321P polymorphism into CE/J cells caused a higher G1 arrest. The pattern of OVCA1 expression also changed from being diffuse throughout the cytoplasm in 129/Sv cells to being punctuate in the cytoplasm of CE/J cells. Tp53+/− abnormal embryos had more proliferating cells than normal embryos, but no obvious difference in differentiated neuronal cells. Tp53−/− small embryos had less differentiated neuronal cells and proliferating cells than normal embryos. Thus, a polymorphism of Ovca1, combined with Mop1, genetically modifies embryonic lethality in Tp53 deficient mice.