Biomedical subjects
P Stambrook
Publications and source records attributed to P Stambrook.
Plk3 functionally links DNA damage to cell cycle arrest and apoptosis at least in part via the p53 pathway.
Polo-like kinase 3 (Plk3, previously termed Prk) contributes to regulation of M phase of the cell cycle (Ouyang, B., Pan, H., Lu, L., Li, J., Stambrook, P., Li, B., and Dai, W. (1997) J. Biol. Chem. 272, 28646-28651). Plk3 physically interacts with Cdc25C and phosphorylates this protein phosphatase predominantly on serine 216 (Ouyang, B., Li, W., Pan, H., Meadows, J., Hoffmann, I., and Dai, W. (1999) Oncogene 18, 6029-6036), suggesting that the role of Plk3 in mitosis is mediated, at least in part, through direct regulation of Cdc25C. Here we show that ectopic expression of a kinase-active Plk3 (Plk3-A) induced apoptosis. In response to DNA damage, the kinase activity of Plk3 was rapidly increased in an ATM-dependent manner, whereas that of Plk1 was markedly inhibited. Recombinant Plk3 phosphorylated in vitro a glutathione S-transferase fusion protein containing p53, but not glutathione S-transferase alone. Recombinant Plk1 also phosphorylated p53 but on residues that differed from those targeted by Plk3. Co-immunoprecipitation and pull-down assays demonstrated that Plk3 physically interacted with p53 and that this interaction was enhanced upon DNA damage. In vitro kinase assays followed by immunoblotting showed that serine 20 of p53 was a target of Plk3. Furthermore, expression of a kinase-defective Plk3 mutant (Plk3(K52R)) resulted in significant reduction of p53 phosphorylation on serine 20, which was correlated with a decrease in the expression of p21 and with a concomitant increase in cell proliferation. These results strongly suggest that Plk3 functionally links DNA damage to cell cycle arrest and apoptosis via the p53 pathway.
PRK, a cell cycle gene localized to 8p21, is downregulated in head and neck cancer.
The human PRK gene encodes a protein serine/threonine kinase of the polo family and plays an essential role in regulating meiosis and mitosis. We have previously shown that PRK expression is downregulated in a significant fraction of lung carcinomas. Our current studies reveal that PRK mRNA expression is downregulated in a majority (26 out of 35 patients) of primary head and neck squamous-cell carcinomas (HNSCC) compared with adjacent uninvolved tissues from the same patients, regardless of stage. In addition, PRK transcripts were undetectable in one of the two HNSCC cell lines analyzed. Ectopic expression of PRK, but not a PRK deletion construct, in transformed A549 fibroblast cells suppresses their proliferation. Furthermore, fluorescence in situ hybridization analyses show that the PRK gene localizes to chromosome band 8p21, a region that exhibits a high frequency of loss of heterozygosity in a variety of human cancers, including head and neck cancers, and that is proposed to contain two putative tumor suppressor genes. Considering that PRK plays an important role in the regulation of the G2/M transition and cell cycle progression, our current studies suggest that deregulated expression of PRK may contribute to tumor development. Genes Chromosomes Cancer 27:332-336, 2000.
MMP and TIMP gene expression in head and neck squamous cell carcinomas and adjacent tissues.
OBJECTIVE: To compare the frequency of gene expression of matrix metalloproteinases (MMP) stromelysins -1, -2 and -3 (MMP-3, -10, and -11), matrilysin (MMP-7), MTI-MMP (MMP-14), and of TIMPs (Tissue Inhibitors of MMPs) -1, -2, -3 and -4 in head and neck squamous cell carcinomas with those of matched adjacent normal tissues. MATERIALS AND METHODS: The present study included 20 surgically removed head and neck squamous cell carcinomas, seven of which were accompanied by matched adjacent oral mucosa excised from the border of the specimens outside the tumor area. RNA isolated from tumors and control samples was subjected to RT-PCR using primers specific for MMP-3, -7, -10, -11 and -14 and for TIMPs -1, -2, -3, and -4. RESULTS: Our findings demonstrate that each of the five MMP genes studied were expressed in essentially all the tumors, while the adjacent marginal tissue samples showed a more varied picture: while stromelysin-3 was located to a majority of the marginal samples, matrilysin was expressed in four of seven adjacent samples, stromelysin-1 and MTI-MMP genes were each expressed in three of these samples, and stromelysin-2 transcript was only expressed in two marginal tissue samples. Whereas TIMP-1 and TIMP-2 transcripts were identified in all tumor and adjacent tissue samples studied, TIMP-3 was expressed, albeit often at low levels, in 17 of 20 tumor samples but only in three of seven adjacent tissues. The novel TIMP-4 gene was not expressed at all. CONCLUSIONS: Specific MMP (MMP-3, -7, -10, -14) and TIMP-3 transcripts observed in head and neck squamous cell carcinomas compared to their frequency in specimens of matching tissues provide important information about expression of extracellular matrix degrading enzymes and their tissue inhibitors in head and neck carcinomas.
Human Prk is a conserved protein serine/threonine kinase involved in regulating M phase functions.
Human prk encodes a novel protein serine/threonine kinase capable of strongly phosphorylating casein but not histone H1 in vitro. prk expression is tightly regulated at various levels during different stages of the cell cycle in lung fibroblasts. The Prk kinase activity is relatively low during mitosis, G1, and G1/S, and peaks during late S and G2 stages of the cell cycle. Recombinant human Prk expressed through the baculoviral vector system is capable of phosphorylating Cdc25C, a positive regulator for the G2/M transition. Human prk shares significant sequence homology with Saccharomyces cerevisiae CDC5 and Drosophila melanogaster polo, both of which are essential for mitosis and meiosis. Full-length prk transcripts greatly potentiate progesterone-induced meiotic maturation of Xenopus laevis oocytes. On the other hand, antisense prk transcripts significantly delay and reduce the rate of oocyte maturation. When expressed in a CDC5 mutant strain of S. cerevisiae, human Prk, but not a deletional mutant protein, fully rescues the temperature-sensitive phenotype of the budding yeast. Taken together, prk may represent a new protein kinase, playing an important role in regulating the onset and/or progression of mitosis in mammalian cells.
Prognostic indicators for squamous cell carcinoma of the oral cavity: a clinicopathologic correlation.
Fifty-three patients with T1 squamous cell cancer of the floor of mouth and ventral surface of the tongue with a known clinical outcome were retrospectively analyzed and arbitrarily divided into "aggressive" and "nonaggressive" groups based on their clinical behavior. Various host and tumor factors were then evaluated in an attempt to determine whether the tumor behavior could have been predicted. The paraffin-embedded tumor specimens were evaluated for tumor differentiation, tumor thickness and tumor invasion, microvessel density, and p53 expression. In addition, a composite morphologic grading score was obtained by combining cell differentiation, nuclear polymorphism, mitosis activity, depth of infiltration, type of infiltration, and lymphatic infiltration. No single technique appeared capable of identifying "aggressive" behavior, although possibly an evaluation of composite factors might show promise in the future.
Mitotic and post mitotic consequences of genomic instability induced by oncogenic Ha-ras.
Induced expression of a mutant human Ha-ras oncogene in NIH3T3 cells leads to the rapid production of multicentric chromosomes, acentric chromosome fragments, double minute chromosomes, increased heteroploidy, and increased capacity to undergo gene amplification. In this study we have used fluorescent-in-situ hybridization (FISH) to demonstrate that induction of the Ha-ras oncogene also leads to disruption of the mitotic machinery, resulting in aberrant mitoses and abnormal daughter cells. Cells induced to express an oncogenic Ha-ras transgene accumulate chromosomes that lag outside of the rest of the chromosomal architecture, chromosomes that form bridges between daughter nuclei at anaphase, and that form micronuclei. Many of these mitotic aberrations contain structurally abnormal chromosomes. These ras-induced changes were suppressed by the introduction of a gene encoding the dominant negative effector of ras, raf 301. Expression of raf301 in cells induced to express Ha-ras reduced the level of growth in soft agar, chromosome aberrations, mitotic aberrations, and frequency of gene amplification. These data provide evidence for an association between Ha-ras induced transformation, chromosome aberrations and gene amplification. Furthermore they offer insight into how the cell responds to the formation of aberrant chromosomes, and how disrupting chromosomal architecture could lead to further imbalances in the distribution of genetic material between daughter cells.
Hypoxic activation of nuclear factor-kappa B is mediated by a Ras and Raf signaling pathway and does not involve MAP kinase (ERK1 or ERK2).
We have previously shown that hypoxia causes the activation of nuclear factor-kappa B (NF-kappa B), and the phosphorylation of its inhibitory subunit, I kappa B alpha, on tyrosine residues. With the use of dominant negative mutants of Ha-Ras and Raf-1, we investigated some of the early signaling events leading to the activation of NF-kappa B by hypoxia. Both dominant negative alleles of Ha-Ras and Raf-1 inhibited NF-kappa B induction by hypoxia, suggesting that the hypoxia-induced pathway of NF-kappa B induction is dependent on Ras and Raf-1 kinase activity. Furthermore, although conditions of low oxygen can also activate mitogen-activated protein kinases (ERK1 and ERK2), these kinases do not appear to be involved in regulating NF-kappa B by low oxygen conditions, as dominant negative mutants of mitogen-activated protein kinase do not inhibit NF-kappa B activation by hypoxia. Since Ras and Raf-1 have been previously shown to work downstream from membrane-associated tyrosine kinases such as Src, we determined if the Src membrane-associated kinase was also activated by low oxygen conditions. We detected an increase in Src proto-oncogene activity within 15-30 min of cellular exposure to hypoxia. We postulate that Src activation by hypoxia may be one of the earliest events that precedes Ras activation in the signaling cascade which ultimately leads to the phosphorylation and dissociation of the inhibitory subunit of NF-kappa B, I kappa B alpha.
Detection of P-glycoprotein in squamous cell carcinomas of the head and neck.
One established mechanism of multidrug resistance is elevated expression of P-glycoprotein. The expression of P-glycoprotein by immunohistochemistry was examined in squamous cell carcinomas of the head and neck using a newly developed monoclonal antibody, UIC2, which is specific to the human MDR1 gene product and recognizes an external epitope of the protein. P-glycoprotein was detected in 60% of the samples. MDR1 expression was compared with clinical response to chemotherapy in eight patients who received MDR1-dependent drugs and response was accurately predicted in seven (89%) of eight patients. Positive P-glycoprotein staining correlated with the degree of tumor differentiation, but not with other clinical factors. Therefore, analyzing the expression of P-glycoprotein may play a role when planning chemotherapeutic regimens for patients with head and neck cancer and may be an additional prognostic and diagnostic tool in these patients.
Identification of an acidic fibroblast growth factor-like activity in a mesoblastic nephroma.
An acidic fibroblast growth factor-like activity was detected in a primary mesoblastic nephroma. Identification was based upon Northern blot analysis of renal tumor RNA and by a biochemical characterization of the growth factor activity. The activity extracted from the tumor stimulated DNA synthesis in both mouse fibroblasts and bovine endothelial cells. This activity was heat-sensitive and enhanced in the presence of heparin. The extract activity bound to immobilized immobilized heparin, eluting at 1.2 M NaCl, and cross-reacted with antiserum to recombinant acidic fibroblast growth factor. We also present evidence for acidic fibroblast growth factor-like molecules in normal immature human kidney, although the amount of the activity detected is less than that in the mesoblastic nephroma. These data suggest that acidic fibroblast growth factor-like activity is present in the developing normal human kidney, and that some renal tumors can continue to express a similar activity.
Size classes of replication units in DNA from sea urchin embryos.
Sea urchin DNA containing replication structures was isolated from two to four cell stage and blastula stage embryos, and examined by electron microscopy. In addition to the expected eye forms, we also observed molecules with large internal single-stranded gaps. Such structures were not present in DNA devoid of replicating molecules such as that isolated from sea urchin sperm. When the size of eye forms and interbubble distances between the two stages were compared, there was no detectable difference. In both stages, we observed two distinct size classes of bubbles and of interbubble distances. In the case of bubble sizes, the smaller size class was comprised of clustered microbubbles that ranged from 200 base pairs to 1 Kilobase (kb) with a mean of 432 base pairs. The large eye forms measured 1--35 kb with a mean of 6.8 kb. Interbubble distances also yielded two distinct populations, with the smaller class ranging from 400 base pairs to 2.3 kb (mean = 1.1 kb) and the larger population ranging from 2.8 to 36 kb (mean = 10.9 kb). Although other possibilities cannot be entirely excluded, the data support the contention that a substantial fraction of the larger eye-form population arises from the fusion of the clustered microbubbles.
Error frequency in 5S RNA from cells grown in 5-bromodeoxyuridine.
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Molecular genetics of malignant insulinoma.
Malignant insulinoma is an rare form of cancer with poor prognosis and a reported 5-year survival of 35%. Relatively little is known about the etiology of this disease or of the oncogenes and tumor suppressor genes that participate in its genesis and progression. To address this issue, several protooncogenes, including K-ras, N-ras, erbB-2, erbB-3,c-myc, c-fos, c-jun were examined. Also analyzed was the expression of the growth factors TGF-alpha, EGF, and insulin as well as the EGF receptor (EGF-R), p53 and the putative anti-metastasis gene nm23-H1. These were examined in malignant insulinomas, benign insulinomas, pancreatic B cell hyperplasias and in normal endocrine pancreas. Normal endocrine pancreas showed moderate immunoreaction for c-myc and a strong reaction for insulin. All other parameters were negative. Benign pancreatic B cell hyperplasias were slightly or moderately positive for N-ras and TGF-alpha, and were weakly positive for EGF-R. They were strongly positive for c-myc and insulin. In malignant insulinomas there was strong immunoreaction for c-myc, TGF-alpha, N-ras, K-ras and p53. Insulin reaction was moderate or strong. Molecular genetic studies have been performed for the presence of activating point mutations in codon 12 of the c-K-ras oncogene. Mutations were detected using primer-mediated, mutant-enriched, polymerase chain reaction-restriction fragment length polymorphism analysis and were further characterized by allele-specific oligonucleotide hybridization. Four out of six patients with malignant insulinoma and two out of eight patients with benign insulinoma harbored K-ras point mutations at codon 12. All patients with mutated K-ras oncogene also had elevated levels of p53 protein as well as c-myc and TGF-alpha. In one extremely malignant case we found concomitant mutation at codon 12 of K-ras and codon 61 of the N-ras gene. Our data are consistent with the idea that malignant progression is accompanied by the progressive accumulation of multiple genetic lesions and suggest that activation of myc, TGF-alpha and ras genes may be early events in the development of insulinoma.