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Biomedical subjects

R J Muschel

Publications and source records attributed to R J Muschel.

At least 37 records · Page 2Linked to original sources
PubMed1998

Radiosensitization and apoptosis.

The toxicity of radiation to living tissues was discovered soon after the discovery of radioactivity itself and this toxicity is the basis for cancer therapy with radiation. Although this mode of therapy is often effective, its success is far from assured. One major difficulty in the implementation of radiotherapy is that normal tissues are also sensitive to killing by radiation so that treatment is often limited by the tolerance of normal tissues for radiation. Thus methods that sensitize tumor cells while sparing normal tissues could potentially lead to greater success with radiation as a therapy. Oncogenes are frequently altered in tumors, but are not in normal tissue making them potential targets for altering radiosensitivity and apoptosis in tumors.

Animals↗
PubMed1998

Cell cycle-dependent usage of transcriptional start sites. A novel mechanism for regulation of cyclin B1.

Cyclin B1 mRNA is expressed temporally throughout the cell cycle with peak expression in G2 and M phase. Both transcriptional and posttranscriptional controls are important for this cell cycle-dependent regulation of cyclin B1 mRNA. In this study, we observed that cyclin B1 has two major transcripts: (a) a constitutively expressed transcript, and (b) a cell cycle-regulated transcript expressed predominantly during G2-M phase. These different transcripts are due to alternative start sites. The constitutively expressed transcript starts 65 bases upstream from the cell cycle-regulated message. Changes in mRNA stability did not appear to control the expression of the cell cycle-specific transcript, but we were able to identify a 24-base pair region of the cyclin B1 promoter spanning the start site of the cell cycle-regulated transcript that was critical for its cell cycle-regulated promoter activity. This suggests that transcriptional regulation is responsible for controlling the presence of each message. The 24-base pair sequence required for cell cycle regulation was notable for containing the nucleotides GGCT repeated three times. The possibility that these two transcripts might be physiologically distinct was raised when the cell cycle-specific transcript was found to be translated more efficiently in vitro than the constitutively expressed transcript. These results characterize a novel mechanism for the regulation of cyclin B1 throughout the cell cycle that is dependent upon the use of different transcriptional start sites.

Base Sequence↗
PubMed1998

Conversion of a radioresistant phenotype to a more sensitive one by disabling erbB receptor signaling in human cancer cells.

Inhibition of cell growth and transformation can be achieved in transformed glial cells by disabling erbB receptor signaling. However, recent evidence indicates that the induction of apoptosis may underlie successful therapy of human cancers. In these studies, we examined whether disabling oncoproteins of the erbB receptor family would sensitize transformed human glial cells to the induction of genomic damage by gamma-irradiation. Radioresistant human glioblastoma cells in which erbB receptor signaling was inhibited exhibited increased growth arrest and apoptosis in response to DNA damage. Apoptosis was observed after radiation in human glioma cells containing either a wild-type or mutated p53 gene product and suggested that both p53-dependent and -independent mechanisms may be responsible for the more radiosensitive phenotype. Because cells exhibiting increased radiation-induced apoptosis were also capable of growth arrest in serum-deprived conditions and in response to DNA damage, apoptotic cell death was not induced simply as a result of impaired growth arrest pathways. Notably, inhibition of erbB signaling was a more potent stimulus for the induction of apoptosis than prolonged serum deprivation. Proximal receptor interactions between erbB receptor members thus influence cell cycle checkpoint pathways activated in response to DNA damage. Disabling erbB receptors may improve the response to gamma-irradiation and other cytotoxic therapies, and this approach suggests that present anticancer strategies could be optimized.

Apoptosis↗
PubMed1998

Caffeine and staurosporine enhance the cytotoxicity of cisplatin and camptothecin in human brain tumor cell lines.

Caffeine and staurosporine have been shown to attenuate G2 delay produced by DNA-damaging agents and to augment the cytotoxicity of these agents in a number of cell lines in vitro. Studies in rodent brain tumor cell lines suggest that modulation of the G2/M transition may not contribute to the enhanced cytotoxicity produced by caffeine in brain tumor cells. To evaluate the impact of agents that decrease G2 delay on the cytotoxicity of chemotherapy in human brain tumor cells, we examined the ability of caffeine and staurosporine to modulate the G2 delay and cytotoxicity produced by cisplatin (CDDP) and camptothecin (CPT) in U251 glioma and DAOY medulloblastoma cells. Synchronized U251 were incubated with 20 microM CDDP in the presence or absence of 2 mM caffeine. DAOY cells were incubated with 100 nM CPT in the presence or absence of 2 nM staurosporine. Caffeine and staurosporine attenuated G2 delay produced by CDDP and CPT, respectively. Clonogenic assays indicated that continuous exposure to 2 mM caffeine substantially lowered the ID50 and ID90 of CDDP in U251 cells without significantly altering plating efficiency. Twenty-four-hour exposure to 2 nM staurosporine lowered the ID50 and ID90 of CPT in DAOY cells without significantly altering plating efficiency. Evaluation of programmed cell death using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay indicated that one mechanism for synergistic cytotoxicty of caffeine with CDDP and staurosporine with CPT in U251 and DAOY cells, respectively, is to promote apoptosis. These results underscore the importance of understanding regulation of G2/M transition in brain tumor cells. Such an understanding may lead to novel therapies that target G2 check points to augment the efficacy of currently available treatments for brain tumors.

Apoptosis↗
PubMed1998

The G2 block induced by DNA damage: a caffeine-resistant component independent of Cdc25C, MPM-2 phosphorylation, and H1 kinase activity.

Treatment of cells with agents that cause DNA damage often results in a delay in G2. There is convincing evidence showing that inhibition of p34cdc2 kinase activation is involved in the DNA damage-induced G2 delay. In this study, we have demonstrated the existence of an additional pathway, independent of the p34cdc2 kinase activation pathway, that leads to a G2 arrest in etoposide-treated cells. Both the X-ray-induced and the etoposide-induced G2 arrest were associated with inhibition of the p34cdc2 H1 kinase activation pathway as judged by p34cdc2 H1 kinase activity and phosphorylation of cdc25C. Caffeine treatment restored these activities after either of the treatments. However, the etoposide-treated cells did not resume cycling, revealing the presence of an alternative pathway leading to a G2 arrest. To explore the possibility that this additional pathway involved phosphorylation of the MPM-2 epitope that is shared by a large family of mitotic phosphoproteins, we monitored the phosphorylation status of the MPM-2 epitope after DNA damage and after treatment with caffeine. Phosphorylation of the MPM-2 epitope was depressed in both X-ray and etoposide-treated cells, and the depression was reversed by caffeine in both cases. The results indicate that the pathway affecting MPM-2 epitope phosphorylation is involved in the G2 delay caused by DNA damage. However, it is not part of the caffeine-insensitive pathway leading to a G2 block seen in etoposide-treated cells.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Overexpression of a transmembrane isoform of neural cell adhesion molecule alters the invasiveness of rat CNS-1 glioma.

CNS-1 is a highly invasive neural cell adhesion molecule (NCAM)-positive rat glioma that exhibits similarities in its pattern of infiltration to human gliomas. To investigate whether increasing NCAM expression alters invasive behavior, retroviruses encoding human NCAM 140 and a cytoplasmic truncation of NCAM 140 were used to transduce a population of CNS-1 glioma cells that had a relatively low endogenous level of NCAM. Compared to cells transduced with a control virus, cells overexpressing either intact or truncated human NCAM 140 showed decreased invasion of a reconstituted basal lamina. Changes in growth rate or in key matrix metalloproteinase activities could not account for this result. In a migration assay on type IV collagen, cells exhibited a substrate concentration-dependent increase in the rate of migration; however, overexpression of NCAM 140 or truncated NCAM 140 inhibited motility at higher substrate concentrations. Consistent with these findings was the decreased spread of NCAM 140 overexpressers in vivo following instillation of cells into the right frontal cortex of rat brain. NCAM 140 overexpressers showed considerably more restricted perivascular and periventricular spread than cells transduced with a control virus. However, NCAM-140-overexpressing tumor exhibited a less cohesive pattern of growth near the site of tumor instillation and more individual cell infiltration of brain parenchyma with more pronounced perineuronal satellitosis. The stability of recombinant NCAM expression was confirmed by recovering tumor cells from tumor-bearing animals and measuring NCAM levels by flow cytometry. These observations show that overexpression of NCAM 140 decreases the long-range spread of CNS-1 glioma along basal lamina pathways but enhances local infiltration of neuropil.

Animals↗
PubMed1998

Inhibiting Ras prenylation increases the radiosensitivity of human tumor cell lines with activating mutations of ras oncogenes.

The influence of activated ras oncogenes on the sensitivity of human tumor cells to killing by radiation has been an unresolved question in radiobiology. We have examined this question by measuring the radiation sensitivity of human tumor cell lines with oncogenic mutations in their H- or K-ras genes after treatment with prenyltransferase inhibitors that prevent the posttranslational modification of ras required for its activity. Using two measures of clonogenic survival, we have demonstrated radiosensitization in cell lines with oncogenic H-ras mutations or with oncogenic K-ras mutations when ras processing was inhibited by prenyltransferase inhibitor treatment. In contrast, the inhibition of ras processing in cell lines expressing wild-type ras had no effect on radiation-induced cell death. The prenyltransferase inhibitors themselves inhibited clonogenic survival in some cases, but this inhibition did not correlate with ras mutational status. Although treatment with prenyltransferase inhibitors and radiation resulted in a greater reduction of clonogenicity than either treatment alone in cells with wild-type ras, treatment with both agents had a synergistic effect on cell killing in tumor cells with ras mutations. Our results demonstrate that the inhibition of oncogenic ras activity in human tumor cells can reduce the radiation survival of these cells, suggesting that oncogenic ras can contribute to radiation resistance in human tumors. These results further demonstrate the potential of using prenyltransferase inhibitors in combination with radiotherapy in the treatment of human malignancies.

Blotting, Western↗
PubMed1998

HIV-1 Vpr interacts with a human 34-kDa mov34 homologue, a cellular factor linked to the G2/M phase transition of the mammalian cell cycle.

Several important and possibly interrelated functions have been identified for the HIV-1 accessory gene product Vpr. These include import of the HIV reverse transcription complex into the nucleus of nondividing cells, cellular differentiation including cell cycle arrest at the G2/M phase border, immune suppression, and enhancement of virus replication. We have cloned a candidate Vpr ligand, termed human Vpr interacting protein (hVIP/MOV34), by using a yeast two-hybrid assay. This gene is homologous to a simultaneously identified 34-kDa human mov34 homologue. The MOV34 family includes proteins that function as transcriptional and proteolytic regulators of cell growth and differentiation. We demonstrate direct interactions between the putative ligand hVIP/MOV34 and Vpr in vitro and in vivo. hVIP/MOV34 localizes to the nucleus and appears to function as a component of the cell cycle cascade. We observe an association between the induction of cell cycle arrest at the G2/M phase border by Vpr and a change in the subcellular localization of hVIP/MOV34 from a nuclear to a perinuclear localization. This was further associated with the inhibition of maturation promoting factor-associated histone H1 kinase activity. We conclude that hVIP/MOV34 is involved in the regulation of the cell cycle and a likely cellular cofactor for HIV-1 Vpr.

Amino Acid Sequence↗
PubMed1998

Tumor cell contact mediated transcriptional activation of the fibroblast matrix metalloproteinase-9 gene: involvement of multiple transcription factors including Ets and an alternating purine-pyrimidine repeat.

The 92-kDa type IV collagenase (MMP-9) is a metalloproteinase frequently localized in both tumor stroma and in tumor cells, particularly at the tumor invasion front. To explore the factors regulating transcriptional activation of MMP-9 in stromal cells, we used a model system in which fibroblast MMP-9 expression can be upregulated by cell-cell contact with metastatic transformed rat embryo cells. Using transient transfection of reporter gene constructs containing 5'-deleted or mutated MMP-9 promoter fragments, as well as electrophoretic mobility shift assays, the upstream NFkappaB, SP-1, and Ets sites and the downstream AP-1 site and retinoblastoma binding element were shown to be necessary for basal transcriptional activity of fibroblast MMP-9. In contrast only Ets or SP-1 appeared to be involved in contact-mediated induction of MMP-9. Mutation of the upstream AP-1 site increased both basal and contact-stimulated promoter activation. Deletion of the alternating purine-pyrimidine repeat in the downstream promoter decreased transcriptional activity. Together these findings suggest that Ets and SP-1 are the central transcriptional activators of MMP-9 gene expression in fibroblasts specifically responding to tumor cell contact, and that promoter conformation may regulate MMP-9 expression.

Animals↗
PubMed1998

Requirement for matrix metalloproteinase-9 (gelatinase B) expression in metastasis by murine prostate carcinoma.

Although a number of effective therapies are available for localized prostate cancer, metastatic prostate cancer is difficult to treat and impossible to cure. Identification of the gene products that enable a prostatic carcinoma cell to metastasize should facilitate an understanding of the processes leading to metastasis. To characterize the contribution of matrix metalloproteinase-9 (MMP-9, gelatinase B or the 92-kd type IV gelatinase/collagenase) to the development of metastasis in prostate cancer, we reduced MMP-9 expression in metastatic murine prostatic carcinoma cells using a ribozyme. The ribozyme transfected cells had lower basal levels of MMP-9 as well as decreased levels after stimulation by transforming growth factor-beta or phorbol 12-myristate 13-acetate when compared with the parental cells or with control transfectants. The cells with down-regulated MMP-9 were unable to form lung colonies in the experimental metastasis assay, whereas the controls and parental cells readily formed metastases. All cell types readily formed tumors after injection and down-regulation of MMP-9 did not adversely affect the rate of tumor growth. Thus, MMP-9 expression is required for hematogenous metastasis in a murine prostate model system raising the possibility that it may play an equivalent role in human prostate cancer.

Animals↗
PubMed1998

Radiation and the G2 phase of the cell cycle.

Exposure of mammalian cells to X rays results in prolongation of the cell cycle, including delays or arrests in G1, S and G2 phase. While G1-phase arrest occurs only in cells with wild-type p53 function, a G2-phase delay occurs in all cells regardless of p53 status. In this review, we summarize what is known about cell cycle progression through G2 and M phase and discuss the experimental findings that implicate different mechanisms in the G2-phase delay. Finally, we consider the possibility that G2-phase arrest plays a role in cell survival after irradiation.

Animals↗
PubMed1997

Transcriptional activation of the matrix metalloproteinase-9 gene in an H-ras and v-myc transformed rat embryo cell line.

The 92 kd type IV collagenase/gelatinase (MMP-9) is important in mediating basement membrane and extracellular matrix degradation in metastasis. Because MMP-9 is made in tumor cells, but not in quiescent normal cells, we wished to identify the transcriptional elements responsible for its synthesis in tumor cells. We chose to characterize transcriptional regulation of the MMP-9 gene in a highly metastatic H-ras and v-myc transformed rat embryo cell line which overexpresses MMP-9. Using transient transfection of reporter gene constructs containing either 5'-deleted or mutated MMP-9 promoter fragments, as well as electrophoretic mobility shift assays, we have demonstrated that multiple transcription factor consensus binding motifs in the promoter, including those for NFkappaB, SP-1, Ets, AP-1, and a retinoblastoma binding element, participate in transcriptional regulation of MMP-9 expression in this cell line. Also, deletion of an alternating purine-pyrimidine tract in the downstream promoter was found to decrease transcriptional activity, suggesting that promoter conformation may be important in MMP-9 regulation. Thus multiple pathways leading to activation of NFkappaB, SP-1, Ets, AP-1, and retinoblastoma binding factors in tumor cells all may contribute to MMP-9 transcription and hence to metastasis.

Animals↗
PubMed1997

Cyclin B1 availability is a rate-limiting component of the radiation-induced G2 delay in HeLa cells.

Irradiation of tumor cells results in a G2 delay, which has been postulated to allow DNA repair and cell survival. The G2 delay after irradiation is marked in HeLa and other cells by delayed expression of cyclin B1. To test whether this depression of cyclin B1 contributes to the G2 delay, we induced cyclin B1 expression in irradiated HeLa cells using a dexamethasone-inducible promoter. Induction of cyclin B1 after radiation abrogated the G2 delay by approximately doubling the rate at which the cells reentered mitosis, whereas dexamethasone itself had no effect. However, overexpression of cyclin B1 did not eliminate the G2 delay in irradiated cells. In unirradiated cells, overexpression of cyclin B1 had no effect on cell cycle progression. Confirmation that reduction of cyclin B1 levels would prolong G2 was provided using antisense oligonucleotides to cyclin B1. These results demonstrate that cyclin B1 levels control the length of the G2 delay following irradiation in HeLa cells but do not exclude additional mechanisms controlling the mitotic delay after irradiation.

Biomarkers↗
PubMed1997

Potential molecular targets for manipulating the radiation response.

Recent advances in our understanding of the molecular events that occur following ionizing radiation leading to DNA damage and repair, apoptosis, and cell-cycle arrests suggest new ways in which the radiation response might be manipulated. Specific targets which, if inactivated, might increase radiosensitivity include Ras, which has been implicated in the radioresistant phenotype, and components of DNA-dependent protein kinase or other molecules involved in the recognition or repair of DNA damage. In some tumors, apoptosis is an important mode of cell death following radiation, so agents that promote this may prove useful therapeutically. Conversely, side effects may result from radiation-induced apoptosis of normal tissues: for example, pneumonitis following the destruction of endothelial cells in the pulmonary vasculature. Therefore, decreasing apoptosis in these tissues may reduce late effects. It may also be possible to prevent late effects such as fibrosis by blocking the induction of certain genes such as transforming growth factor beta. Cell-cycle regulation is another area that could be manipulated to increase radiosensitivity. There is evidence that the G2 delay following radiation is important in protecting cells from death. Abolition of this delay may increase radiosensitivity, especially in cells with mutant p53 that have lost the G1 checkpoint.

Animals↗
PubMed1997

The molecular regulation of apoptosis and implications for radiation oncology.

One of the major goals of cancer research is to identify and understand the causes of cellular proliferation. The role of cell death, or lack thereof, in carcinogenesis, tumour growth, metastatic spread and response to treatment has been largely overlooked even though the morphology of apoptosis (programmed cell death) was clearly described over 20 years ago, and its importance in cancer speculated on at that time. Over the last 5 years, however, an explosion of research has focused on delineating the molecular components of the apoptotic pathways and examining the role of apoptosis in a tumour's growth and response to treatment. This review highlights the aspects of apoptosis most relevant to radiation oncologists and radiobiologists. The apoptotic pathways will be described, with attention to the stimuli that initiate apoptosis, the oncogenes and tumour suppressor genes that mediate apoptosis, and the effector enzymes (proteases and endonucleases) responsible for the execution of apoptosis. In addition, we review the effect of classically described radiobiology cell survival parameters-cell cycle stage, dose rate, linear energy transfer, oxygen, total dose, and fractionation-on radiation induced apoptosis.

Animals↗
PubMed1997

Cyclin A message stability varies with the cell cycle.

Progression through the cell cycle in somatic eukaryotic cells is regulated by variations in the levels of cyclin proteins. These protein levels are in turn regulated by cyclical oscillations in their mRNA levels. We show here that regulation of RNA stability plays a role in the mechanisms underlying cell cycle progression. Both cyclin A and B1 messages are expressed at high levels in G2-M and at low levels in early G1. The half-lives of their messages mirror this pattern, long in G2-M (>8 h) and short in early G1 (1-2 h). However, there is evidence of specificity to these changes, because the cyclin A message becomes stable at the G1-S boundary, whereas the cyclin B1 message is unstable until later in S phase. Furthermore, although cyclin B1 mRNA levels are lowered after irradiation because of enhanced instability, cyclin A mRNA levels and message stability are unaffected by irradiation. Additional evidence of specificity was found in an analysis of cyclin E mRNA stability, which remains constant through the cell cycle, although the cyclin E message displays cell cycle-dependent changes in expression. These studies suggest that specific alterations in RNA stability are an important component in regulating the expression of cyclins A and B1 and hence in controlling the cell cycle.

Blotting, Northern↗