PubMed Health⌕ Search

Biomedical subjects

P Kirschmeier

Publications and source records attributed to P Kirschmeier.

17 recordsLinked to original sources
PubMed1996

Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy.

The growth factor receptor-bound protein-2 (Grb-2) is an adaptor protein that mediates signal transduction pathways. Chemical shift assignments were obtained for the SH2 domain of Grb2 by heteronuclear NMR spectroscopy, employing the uniformly 13C-/15N-enriched protein as well as the protein containing selectively 15N-enriched amino acids. Using the Chemical Shift Index (CSI) method, the chemical shift indices of four nuclei, 1H alpha, 13C alpha, 13C beta and 13CO, were used to derive the secondary structure of the protein. Nuclear Overhauser enhancements (NOEs) were then employed to confirm the secondary structure. The CSI results were compared to the secondary structural elements predicted for the Grb2 SH2 domain from a sequence alignment [Lee et al. (1994) Structure, 2, 423-438]. The core structure of the SH2 domain contains an antiparallel beta-sheet and two alpha-helices. In general, the secondary structural elements determined from the CSI method agree well with those predicted from the sequence alignment.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Identification and characterization of zinc binding sites in protein kinase C.

Metal ion coordination in the regulatory domain of protein kinase C (PKC) is suggested by the conservation of six cysteines and two histidines in two homologous regions found therein. By monitoring x-ray fluorescence from a purified sample of rat PKC beta I overexpressed in insect cells, direct evidence has been obtained that PKC beta I tightly binds four zinc ions (Zn2+) per molecule. Extended x-ray absorption fine structure (EXAFS) data are best fit by an average Zn2+ coordination of one nitrogen and three sulfur atoms. Of the plausible Zn2+ coordination models, only those featuring nonbridged Zn2+ sites accommodate the EXAFS data and all of the conserved potential ligands.

Absorptiometry, Photon↗
PubMed1988

Overproduction of protein kinase C causes disordered growth control in rat fibroblasts.

We have generated a series of rat fibroblast cell lines that stably overexpress a full-length cDNA encoding the beta 1 form of protein kinase C (PKC). These cell lines contain a 20- to 53-fold increase in PKC activity and exhibit dramatically enhanced morphologic changes following exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). They grow to a high saturation density in monolayer cultures and, when maintained at postconfluence, develop small, dense foci. In contrast to control cells, which display complete anchorage dependence, PKC-overproducing cells form small colonies in soft agar in the absence of TPA and large colonies in the presence of TPA. Thus, the mere overproduction of a single form of PKC is sufficient to confer multiple growth abnormalities in rat fibroblasts. These results provide direct evidence that PKC plays a critical role in growth control and that it mediates several of the cellular effects of the phorbol ester tumor promoters. They also suggest that the activation of PKC may be of central importance in the process of multistage carcinogenesis.

Amino Acid Sequence↗
PubMed1987

Isolation of cDNA clones encoding protein kinase C: evidence for a protein kinase C-related gene family.

We have isolated cDNA clones encoding protein kinase C by using a 53-base-pair synthetic oligonucleotide probe corresponding to a peptide that we obtained from the rat brain enzyme. We also have isolated several closely related clones using the same oligonucleotide probe. Nucleotide sequence analysis of one of the protein kinase C clones, RP41, identifies a 224-amino-acid carboxyl-terminal region with approximately equal to 40% homology to the carboxyl-terminal catalytic domains of both the cAMP-dependent and cGMP-dependent protein kinases. The levels of mRNA homologous to RP41 are very high in brain, whereas much lower levels are present in heart and liver. Nucleotide sequence analysis of a second cDNA clone, RP16, identifies a deduced amino acid sequence that shares 65% homology with the corresponding region of the protein kinase C clone RP41. The levels of mRNA corresponding to RP16 are also high in rat brain, but the transcript sizes and tissue-specific expression patterns differ from those of RP41. These and additional results provide evidence that the gene encoding protein kinase C is a member of a novel serine/threonine protein kinase multigene family.

Animals↗
PubMed1986

Studies on the mechanism of action of protein kinase C and the isolation of molecular clones encoding the enzyme.

Protein kinase C (PKC) plays an important role in signal transduction and the action of phorbol ester tumor promoters, and it is of interest to isolate the coding sequence of this enzyme. Using a 53-base pair synthetic oligonucleotide probe that corresponds to an 18-amino acid peptide obtained from rat brain PKC, we have isolated rat brain cDNA clones corresponding to PKC. We have also isolated several closely related clones. Partial nucleotide sequence analysis of one of the PKC clones (RP41) identifies a 224-amino acid region with approximately 40% homology to the carboxy terminal and catalytic domains of both the cAMP-dependent and cGMP-dependent protein kinases. The levels of mRNA homologous to RP41 are very high in brain; very low but detectable levels are present in heart and liver. A second cDNA (RP16) was only partially sequenced, and based on its predicted amino acid sequence, it shares 65% homology with the corresponding region of the PKC clone RP41. The levels of mRNA corresponding to RP16 are also highest in rat brain, but they are of a different size than those detected with RP41. These and additional results indicate that the gene for the enzyme PKC shares considerable homology with other protein kinases and that PKC itself may belong to a new multigene family. The availability of these cDNA clones should greatly facilitate further studies on the role of PKC in growth control, differentiation, and multistage carcinogenesis.

Amino Acid Sequence↗
PubMed1985

Expression of long terminal repeat (LTR) sequences in carcinogen-induced murine skin carcinomas.

RNA sequences homologous to the Long Terminal Repeat (LTR) sequence of Moloney Murine Leukemia Virus proviral DNA are expressed in murine squamous cell carcinomas of the skin induced by chemical carcinogens. These transcripts range in size from 8.2 to less than 2.4 kb but their size profile varies between individual tumors. These RNAs are not detected in the poly A+ RNA fraction obtained from the epidermis of control mice or carcinogen induced skin papillomas. The poly A+ RNAs from the livers and spleens of some of the mice with skin carcinomas also revealed LTR related sequences, whereas these RNAs were not detected in the livers and spleens of control mice or of carcinogen-treated mice that did not develop carcinomas. Thus, chemical carcinogenesis in mouse skin is associated with constitutive expression of endogenous retrovirus related sequences in the carcinomas as well as in certain apparently normal host tissues.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1985

Mechanisms of multistage chemical carcinogenesis and their relevance to respiratory tract cancer.

The evolution of a fully malignant tumor is a multistep process resulting from the action of multiple factors, both environmental and endogenous, and involves alterations in the function of multiple cellular genes. Chemical carcinogens that initiate this process appear to do so by damaging cellular DNA. In addition to producing simple point mutations, this damage appears to induce the synthesis of a transacting factor that can induce asynchronous DNA replication. This response may result in gene amplification and/or gene rearrangement. This phenomenon may also play a role in synergistic interactions between chemicals and viruses in the causation of certain cancers. The primary target of the tumor promoters TPA, teleocidin, and aplysiatoxin appears to be cell membranes. All three of these agents act, at least in part by, enhancing the activity of the phospholipid-dependent enzyme PKC. We have proposed a stereochemical model to explain the interaction of these amphiphilic compounds with the PKC system. We have found that TPA and teleocidin markedly enhance the transformation of C3H10T1/2 mouse fibroblasts when these cells are transfected with the cloned H-ras human bladder cancer oncogene. Thus, tumor promoters can act synergistically with an activated oncogene to enhance cell transformation. Furthermore, carcinogen-transformed rodent cells display aberrations in the expression of various endogenous retrovirus-related sequences. Activation of some of these sequences may lead to insertion mutations and further aberrations in gene expression. These findings are discussed in terms of a multistep model that involves progressive changes in cellular oncogenes and aberrations in the function of DNA transcription enhancer sequences. It will be of interest to determine to what extent these concepts apply to the etiology of cancers of the respiratory tract.

Animals↗
PubMed1984

Cellular targets and host genes in multistage carcinogenesis.

Recent studies indicate that although cellular DNA is the critical target in the action of initiating carcinogens, specific membrane-associated receptors mediate the actions of certain tumor promoters. A stereochemical model is presented to explain how three different types of tumor promoters (phorbol esters, indole alkaloids, and polyacetates) can interact with the same class of cellular receptors. Multistage chemical carcinogenesis might involve progressive alterations in the expression of cellular DNA sequences homologous to oncogenes and regulatory sequences in certain retroviruses. We found that the oncogene c-mos is not rearranged or expressed in a series of carcinogen-transformed murine C3H 10T112 cells. These cells do express, however, a unique set of poly(A)+ RNAs that contain sequences homologous to the Moloney leukemia virus long terminal repeat sequence. Studies are in progress to determine the significance of this finding with respect to the carcinogenic process.

Animals↗
PubMed1984

Effects of 5-azacytidine on methylation and expression of specific DNA sequences in C3H 10T1/2 cells.

The present study indicates that the transient exposure of C3H 10T1/2 mouse embryo fibroblasts to 5-azacytidine leads to extensive loss of methylation of the protooncogene c-mos and the beta-globin locus at the cell population level and in at least 40 isolated subclones. These changes persisted, even when the cells were serially passaged for many generations without further exposure to the drug. Even though the amount of demethylation, assessed through differential digestion by the restriction enzymes HpaII and MspI, was quite extensive, neither locus was transcribed at a detectable level. This nonselective analysis suggests, therefore, that loss of DNA methylation is not sufficient per se to induce the expression of certain loci. Presumably, the expression of these loci requires additional factors, some of which may be related to cell lineage and differentiation.

Animals↗
PubMed1983

Benzo[a]pyrene induction of extrachromosomal viral DNA synthesis in rat cells transformed by polyoma virus.

The effect of the carcinogen benzo[a]pyrene (BP) on the production of specific extrachromosomal DNAs has been studied in transformed rat cells containing integrated polyoma virus DNA. Exposure of cells previously transformed by the polyoma virus mutant ts-a, which codes for a temperature sensitive T antigen, to a non-toxic dose of BP (0.25 micrograms/ml) enhanced the production of free polyoma DNA at the population level. This occurred at 33 degrees C (the permissive temperature) but not at 39 degrees C, indicating that the BP effect was dependent on the function of the T antigen of polyoma virus. In the same cell system, BP did not induce the production of extrachromosomal copies of two endogenous rat retrovirus genomes, the 30S RNA virus and rat leukemia virus. Thus, the effects on integrated polyoma DNA are quite specific and do not reflect a generalized increase in asynchronous DNA replication and excision. Further studies utilizing a carcinogenic metabolite of BP, benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide (anti) (BPDE), on a rat cell line transformed by wild type (WT) polyoma virus demonstrate that the enhancement of polyoma DNA synthesis persists for at least 5 days after a single exposure to BPDE, despite the rapid decay of this compound. In addition, enhanced synthesis of polyoma DNA can be induced by fusion of normal rat fibroblasts previously exposed to BPDE with polyoma transformed rat fibroblasts not exposed to BPDE. It appears, therefore, that the effects we have observed are not due solely to direct carcinogen damage at the level of the integrated polyoma virus DNA, but may instead involve the induction of cellular factor(s) that act indirectly to enhance asynchronous replication of polyoma DNA.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1983

Molecular mechanisms of tumor promotion and multistage carcinogenesis.

Carcinogenesis is a multistep process resulting from a complex interaction between multiple factors, both environmental and exogenous. In contract to initiating agents that act by damaging cellular DNA, the primary targets of the phorbol ester tumor promoters are membrane-associated receptors. We have proposed a stereochemical model to explain the interaction of these amphiphilic molecules, and of teleocidin and aplysiatoxin, with this receptor system. The model is consistent with evidence that a complex between protein kinase C and phospholipid is the actual receptor for these compounds. Recent data we have obtained with a compound present in tung oil, 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (HHPA), and twelve of its congener's (provided by Y. Ito et al.) are also consistent with our stereochemical model. We have studied phorbol ester receptors in a wide variety of tissue culture cell types. Our data, together with other findings, provide evidence for considerable receptor heterogeneity and this may relate to the pleiotropic effects of these compounds. We have found a case of "masked" receptors in a rat liver cell line and shown that it is due to a cell-associated esterase. Normal human melanocyte cultures contain phorbol ester receptors and this is of particular interest since these cells actually require these or related compounds for optimal growth (in collaboration with M. Eisinger). The receptor studies provide clues to how tumor promoters can, via inductive mechanisms, produce alterations in the structure and function of cell membranes. It is not known, however, how in the multistep carcinogenic process promoters enhance the eventual outgrowth of permanently altered tumor cells. We have found that TPA and teleocidin produce a marked enhancement of transformation of C3H 10T1/2 cells induced by transfection with h-ras human bladder cancer oncogene. These and other results are discussed in terms of the role of alterations in cellular oncogenes and transcriptional enhancer sequences during multistage carcinogenesis.

Adenosine Triphosphate↗
PubMed1982

Carcinogen- and radiation-transformed C3H 10T1/2 cells contain RNAs homologous to the long terminal repeat sequence of a murine leukemia virus.

Carcinogen- or radiation-transformed C3H 10T1/2 murine fibroblasts transcribe a set of poly(A)+RNAs that contain sequences homologous to the long terminal repeat (LTR) sequence of Moloney murine sarcoma virus. These LTR-containing RNAs consist of a series of discrete bands ranging in size from about 38 to 18 S. The higher molecular weight molecules (30-38 S) in this set of RNAs also contain sequences homologous to the gag, pol, and env genes of a murine leukemia virus. A 24S RNA contains sequences homologous to the env gene of murine leukemia virus. A 20S and an 18S RNA also share homology with the LTR probe but fail to hybridize to the gag, pol, or env probes or to a probe for the U3 region of the LTR sequence. Thus, the latter transcripts do not appear to arise from a known endogenous murine leukemia virus genome. Although this entire set of RNAs is absent from normal C3H 10T1/2 cells (or is present at an extremely low level), these RNAs are induced by BrdUrd or 5-azacytidine. The presence of these RNAs may provide highly sensitive molecular markers of transformation of murine cells.

Animals↗
PubMed1982

Cellular Moloney murine sarcoma (c-mos) sequences are hypermethylated and transcriptionally silent in normal and transformed rodent cells.

Moloney murine sarcoma virus carries an oncogenic sequence (v-mos) which is homologous to a single copy gene (c-mos) present in the normal cells of several vertebrate species. Because of the possible significance of c-mos sequences in normal development and malignant transformation induced by physical or chemical agents, we have examined the state of integration, methylation, and transcriptional activity of c-mos sequences in a variety of normal rodent tissues, normal cell lines, or cell lines transformed by radiation or chemical carcinogens. DNA-DNA hybridization, utilizing the Southern blotting technique and a plasmid-derived DNA probe representing the v-mos sequence, gave no evidence for rearrangements of the c-mos sequence in the DNAs obtained from these diverse cell types. Parallel studies employing the restriction enzyme isoschizomers HpaII and MspI indicated that in all of these cell types the c-mos sequences were heavily methylated. In addition, analysis of cellular RNAs by blot hybridization with the v-mos probe failed to detect evidence of transcription of the c-mos sequences in any of these cell types. This was in contrast to a Moloney sarcoma virus-transformed cell line in which we found that the integrated v-mos sequence was both undermethylated and extensively transcribed. Thus, it would appear that c-mos sequences do not play a role in the transformation of rodent cells by chemical or physical agents, although the possible role of other endogenous onc sequences remains to be determined.

Animals↗
PubMed1982

The possible role of cellular genes related to retroviruses in the process of chemical and radiation carcinogenesis.

Chemical carcinogens, radiation, and tumor promoters can not introduce new genetic information into cells. They must, therefore, call upon genes already present in the target cell. We have recently explored the possibility that these host genes share homology with specific nucleic acid sequences present in some of the known retroviruses. In one set of experiments we used a cloned DNA probe prepared from the oncogene (v-mos) of Moloney murine sarcoma virus (Mo-MSV) to examine the state of integration, methylation, and transcription of the homologous cellular oncogene (c-mos) in normal murine cells and in murine cells transformed by radiation or chemical carcinogens. We found that in the transformed cells the c-mos sequence has not undergone rearrangement within the host genome and, as in normal cells, this sequence is hypermethylated and transcriptionally silent. These results are in sharp contrast to the situation in cells transformed by Mo-MSV in which the exogenous v-mos sequence is integrated at new sites within the host genome, is undermethylated and is extensively transcribed. In related studies, we have found that another one gene, c-ras (H), also fails to show any evidence for rearrangement in carcinogen or radiation transformed C3H 10T1/2 cells. Since there is evidence that eukaryotic cells contain numerous onc genes our results do not rule out the possibility that onc genes other than c-mos and c-ras (H) may play a role in the transformation of murine cells by chemicals or radiation. Additional studies utilizing probes to other onc genes are required to evaluate this possibility.

Animals↗