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Biomedical subjects

R J Muschel

Publications and source records attributed to R J Muschel.

At least 19 recordsLinked to original sources
PubMed2003

Farnesyltransferase inhibitors as radiation sensitizers.

PURPOSE: The inhibition of activated Ras combined with radiotherapy was identified as a potential method for radiosensitization. MATERIALS AND METHODS: Immunoblotting was used to control for prenylation inhibition of the respective Ras isoforms and for changes in activity of downstream proteins. Clonogenic assays with human and rodent tumour cell lines and transfected cell lines served for the testing of radiosensitivity. Xenograft tumours were treated with farnesyl transferase inhibitors and radiation and assayed for ex vivo plating efficiency, regrowth of tumours and EF5 staining for detection of hypoxia. Concurrent treatment with L-778,123 and radiotherapy was performed in non-small cell lung cancer (NSCLC) and head and neck cancer (HNC) patients. RESULTS: Blocking the prenylation of Ras proteins in cell lines with Ras activated by mutations or receptor signalling resulted in radiation sensitization in in vitro and in vivo. The PI3 kinase downstream pathway was identified as a contributor to Ras-mediated radiation resistance. Additionally, increased oxygenation of xenograft tumours was observed after FTI treatment. Combined treatment in a phase I study was safe and effective in NSCLC and HNC. CONCLUSIONS: Tumour cells with activated Ras were sensitized to radiation. Unravelling the underlying mechanisms promises to lead to even more specific drugs with higher potency and safety.

Alkyl and Aryl Transferases↗
PubMed2001

The Ras radiation resistance pathway.

The critical pathways determining the resistance of tumor cells to ionizing radiation are poorly defined. Because the ras oncogene, a gene activated in many human cancers treated with radiotherapy, can induce increased radioresistance, we have asked which Ras effector pathways are significant in conferring a survival advantage to tumor cells. The phosphoinositide-3-kinase (PI3K) inhibitor LY294002 radiosensitized cells bearing mutant ras oncogenes, but the survival of cells with wild-type ras was not affected. Inhibition of the PI3K downstream target p70S6K by rapamycin, the Raf-MEK-MAPK pathway with PD98059, or the Ras-MEK kinase-p38 pathway with SB203580 had no effect on radiation survival in cells with oncogenic ras. Expression of active PI3K in cells with wild-type ras resulted in increased radiation resistance that could be inhibited by LY294002. These experiments have indicated the importance of PI3K in mediating enhanced radioresistance and have implicated PI3K as a potential target for specific radiosensitization of tumors.

Animals↗
PubMed2001

Paradoxical effects of trichostatin A: inhibition of NF-Y-associated histone acetyltransferase activity, phosphorylation of hGCN5 and downregulation of cyclin A and B1 mRNA.

Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), is widely used to study the role of histone acetylation in gene expression, since genes that use histone acetylation as a means of regulating expression may be up regulated when TSA is added. In this study, however, we show that TSA has an unexpected paradoxical effect leading to inhibition of NF-Y-associated histone acetyl transferase (HAT) activity and phosphorylation of the HAT, hGCN5. TSA treatment of cells resulted in diminished levels of NF-Y-associated HAT activity without changes in NF-Y(A) amount. hGCN5 is one of the HATs known to associate with NF-Y. The association of hGCN5 with NF-Y was not altered by TSA treatment. The enzymatic activity of hGCN5 is known to be inhibited by phosphorylation. TSA treatment of Hela cells resulted in phosphorylation of hGCN5. Exposure of the NF-Y immunoprecipitates from TSA-treated cells to a phosphatase resulted in enhanced HAT activity. We have also shown that the mRNA levels of several genes, cyclin B1 and cyclin A, are downregulated by TSA; these effects do not require protein synthesis and the downregulation of cyclin B1 by TSA occurs through transcription. These results suggest that TSA can have contradictory effects, on one hand stimulating HAT activity in general by inhibition of HDACs, but also resulting in inhibition of NF-Y-associated HAT activity and phosphorylation of hGCN5.

Acetyltransferases↗
PubMed2001

The farnesyltransferase inhibitor L744,832 reduces hypoxia in tumors expressing activated H-ras.

Many tumors contain extensive regions of hypoxia. Because hypoxic cells are markedly more resistant to killing by radiation, repeated attempts have been made to improve the oxygenation of tumors to enhance radiotherapy. We have studied the oxygenation of tumor xenografts in nude mice after treatment with the farnesyltransferase inhibitor L744,832. Hypoxia was assessed by measuring the binding of the hypoxic cell marker pentafluorinated 2-nitroimidazole. We show that xenografts from two tumor cell lines with mutations in H-ras had markedly improved oxygenation after farnesyltransferase treatment. In contrast, xenografts from two tumors without ras mutations had equivalent hypoxia regardless of treatment. The effect on tumor oxygenation could be detected at 3 days and remained after 7 days of treatment. These results indicate that treatment with farnesyltransferase inhibitors can alter the oxygenation of certain tumors and suggest that such treatment might be useful in the radiosensitization of these tumors.

Alkyl and Aryl Transferases↗
PubMed2001

Apoptosis: an early event in metastatic inefficiency.

Whereas large numbers of cells from a primary tumor may gain access to the circulation, few of them will give rise to metastases. The mechanism of elimination of these tumor cells, often termed "metastatic inefficiency," is poorly understood. In this study, we show that apoptosis in the lungs within 1-2 days of introduction of the cells is an important component of metastatic inefficiency. First, we show that death of transformed, metastatic rat embryo cells occurred via apoptosis in the lungs 24-48 h after injection into the circulation. Second, we show that Bcl-2 overexpression in these cells inhibited apoptosis in culture and also conferred resistance to apoptosis in vivo in the lungs 24-48 h after injection. This inhibition of apoptosis led to significantly more macroscopic metastases. Third, comparison between the extent of apoptosis by a poorly metastatic cell line to that by a highly metastatic cell line 24 h after injection in the lungs revealed more apoptosis by the poorly metastatic cell line. These results indicate that apoptosis, which occurs at 24-48 h after hematogenous dissemination in the lungs is an important determinant of metastatic inefficiency. Although prior work has shown an association between apoptosis in culture and metastasis in vivo, this work shows that apoptosis in vivo corresponds to decreased metastasis in vivo.

Animals↗
PubMed2001

Decreased cyclin B1 expression contributes to G2 delay in human brain tumor cells after treatment with camptothecin.

DNA damage produces delayed mitosis (G2/M delay) in proliferating cells, and shortening the delay sensitizes human malignant glioma and medulloblastoma cells to cytotoxic chemotherapy. Although activation of the cyclin-dependent kinase CDC2 mediates G2/M transition in all tumor cells studied to date, regulation of CDC2 varies between tumor types. Persistent hyperphosphorylation of kinase and reduced cyclin expression have been implicated as mediators of treatment-induced G2 delay in different tumor models. To evaluate regulation of G2/M transition in human brain tumors, we studied the expression and/or activity of CDC2 kinase and cyclins A and B1 in U-251 MG and DAOY medulloblastoma cells after their treatment with camptothecin (CPT). Synchronized cells were treated during S phase, then harvested at predetermined intervals for evaluation of cell cycle kinetics, kinase activity mRNA, and protein expression. CPT produced G2 delay associated with decreased CDC2 kinase activity and cyclin B1 expression. Kinase activity was associated with CDC2 bound to cyclin B1, not cyclin A, in both cell lines. Cyclin A mRNA and protein expression were reduced after CPT treatment; however, decreased protein expression was short lived and moderate in the glioma and primitive neuroectodermal tumor/medulloblastoma cells, respectively. We conclude that G2 delay is a common response of brain tumor cells to chemotherapy with topoisomerase I inhibitors and that a mechanism of this delay may be reduced expression of cyclin B1.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Direct evidence for the contribution of activated N-ras and K-ras oncogenes to increased intrinsic radiation resistance in human tumor cell lines.

Transformation with ras oncogenes results in increased radiation sur vival in many but not all cells. In addition, prenyltransferase inhibitors which inhibit ras proteins by blocking posttranslational modification radiosensitize cells with oncogenic ras. These findings suggest that oncogenic ras contributes to intrinsic radiation resistance. However, because introduction of ras oncogenes does not increase radiation survival in all cells and because prenyltransferase inhibitors target molecules other than ras, these studies left the conclusion that ras increases the intrinsic radi ation resistance of tumor cells in doubt. Here we show that genetic inactivation of K- or N-ras oncogenes in human tumor cells (DLD-1 and HT1080, respectively) leads to increased radiosensitivity. Reintroduction of the activated N-ras gene into the HT1080 line, having lost its mutant allele, resulted in increased radiation resistance. This study lends further support to the hypothesis that expression of activated ras can contribute to intrinsic radiation resistance in human tumor cells and extends this finding to the K- and N- members of the ras family. These findings support the development of strategies that target ras for inactivation in the treatment of cancer.

Adenocarcinoma↗
PubMed2000

BRCA1 effects on the cell cycle and the DNA damage response are linked to altered gene expression.

The breast and ovarian cancer susceptibility gene product BRCA1 has been reported to be expressed in a cell cycle-dependent manner; possess transcriptional activity; associate with several proteins, including the p53 tumor suppressor; and play an integral role in certain types of DNA repair. We show here that ectopic expression of BRCA1 using an adenovirus vector (Ad-BRCA1) leads to dephosphorylation of the retinoblastoma protein accompanied by a decrease in cyclin-dependent kinase activity. Flow cytometric analysis on Ad-BRCA1-infected cells revealed a G(1) or G(2) phase accumulation. High density cDNA array screening of colon, lung, and breast cancer cells identified several genes affected by BRCA1 expression in a p53-independent manner, including DNA damage response genes and genes involved in cell cycle control. Notable changes included induction of the GADD45 and GADD153 genes and a reduction in cyclin B1 expression. Therefore, BRCA1 has the potential to modulate the expression of genes and function of proteins involved in cell cycle control and DNA damage response pathways.

Adenoviridae↗
PubMed2000

Matrix metalloproteinase 9 promoter activity is induced coincident with invasion during tumor progression.

Matrix metalloproteinase 9 (MMP-9, also known as gelatinase B or 92-kd Type IV collagenase) is overexpressed in many human and murine cancers. We induced carcinomas in mice carrying a transgene that links the MMP-9 promoter to the reporter ss-galactosidase so that activation of the MMP-9 promoter would be indicated by ss-galactosidase. Mammary carcinomas were induced by mating the MMP-9 promoter reporter transgenic mice with mice carrying a transgene for murine mammary tumor virus promoter linked to polyoma middle T antigen, a transgene that leads to rapid development of mammary tumors in female mice. None of the hyperplastic mammary glands and none of the carcinomas in situ expressed ss-galactosidase. However, all invasive tumors had evidence of ss-galactosidase expression. In addition to the breast carcinomas, a malignant teratoma in a female and a papillary adenocarcinoma in the pelvic region of a male arose and were also ss-galactosidase positive. We also induced skin tumors in the mice with the MMP-9 reporter transgene with 7, 12-dimethylbenz[a]anthracene (DMBA) treatment followed by phorbol 12 myristate 13-acetate (TPA). None of the papillomas or in situ carcinomas showed any ss-galactosidase expression, but expression was seen in invasive carcinoma. Although normal skin epithelial cells did not express ss-galactosidase, we did find staining in a few cells at the duct of the sebaceous gland at the base of the hair follicles. The MMP-9 reporter transgene did not lead to expression in the alveolar macrophages, confirming that additional upstream sequences are required for expression in macrophages. These experiments have revealed that MMP-9 promoter activity is induced coincident with invasion during tumor progression. Furthermore, this indicates that the more proximal upstream elements of the promoter are sufficient for MMP-9 transcription during tumor progression.

Animals↗
PubMed2000

Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.

Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.

Animals↗
PubMed2000

DNA as an important target in radiation-induced apoptosis of MYC and MYC plus RAS transfected rat embryo fibroblasts.

PURPOSE: This study uses a radiation chemistry approach to determine if DNA is an important target for radiation-induced apoptosis of myc (MR4) and myc plus ras (3.7) transfected rat embryo fibroblast cell lines. MATERIALS AND METHODS: The radiation protection efficiency of four thiols was compared with net molecular charge ranging from -1 to +2: mercaptopropionic acid (Z= -1), mercaptoethanol (Z=0), cysteamine (Z= +1), N(2-mercaptoethyl)-1,3-diaminopropane (Z= +2). Protection factors were determined for these thiols against radiation-induced apoptosis (Apoalert assay), mitotic cell death (clonogenic assay) and double-strand break (dsb) induction (pulse field gel electrophoresis) in MR4 and 3.7 cells. Theoretical protection factors for these thiols against dsb induction were also calculated from second-order chemical repair constants for single-strand breaks (ssb) and the concentration of added thiols in MR4 and 3.7 cell lines. RESULTS: The charge-dependent increases observed for measured protection factors against radiation-induced apoptosis did not differ significantly between the two cell lines, nor did they differ significantly from the corresponding increases observed for radiation-induced mitotic cell killing and for induction of dsb. The calculated protection factor for dsb also showed a thiol charge-dependent increase similar to the measured protection factors for all of the other parameters studied. CONCLUSIONS: These results are consistent with the hypothesis that DNA is an important target for radiation-induced apoptosis.

Animals↗
PubMed2000

RAS-Mediated radiation resistance is not linked to MAP kinase activation in two bladder carcinoma cell lines.

The expression of activated RAS oncogenes has been shown to increase radioresistance in a number of cell lines. The pathways by which RAS leads to radioresistance, however, are unknown. RAS activates several signal transduction pathways, with the RAF-MAP2K-MAP kinase pathway perhaps the best studied. MAP kinase has also been shown to be activated by radiation through this pathway. Given the important role of MAP kinase in multiple signaling events, we asked if radioresistance induced by RAS was mediated through the activation of MAPK. Cells of two human bladder carcinoma cell lines were used, one with a mutated oncogenic HRAS (T24) and other with a wild-type HRAS (RT4). The surviving fraction after exposure to 2 Gy of radiation (SF2) for the T24 cell lines was found to be 0.62, whereas that for RT4 cells was 0.40. Treatment with the farnesyl transferase inhibitor (FTI) L744,832, which inhibits RAS processing and activity, decreased the SF2 of T24 cells to 0.29, whereas the SF2 of RT4 cells remained unchanged after FTI treatment, thus demonstrating the importance of RAS activation to the radiosensitivity of cells with mutated RAS. MAP kinase activation was found to be constitutive and dependent on RAS in T24 cells, while it was inducible by radiation and was independent of RAS in RT4 cells. Treatment of both cell lines with the MAP2K inhibitor PD98059 inhibited MAPK activation; however, inhibiting MAPK activation had no effect on radiation survival of T24 or RT4 cells. These data indicate that MAPK activation does not contribute to RAS-induced radioresistance in this system.

Cell Survival↗
PubMed2000

Farnesyltransferase inhibitors potentiate the antitumor effect of radiation on a human tumor xenograft expressing activated HRAS.

Successful radiosensitization requires that tumor cells become more radiosensitive without causing an equivalent reduction in the survival of cells of the surrounding normal tissues. Since tumor cell radiosensitivity can be influenced by RAS oncogene activation, we have hypothesized that inhibition of oncogenic RAS activity would lead to radiosensitization of tumors with activated RAS. We previously showed in tissue culture that prenyltransferase treatment of cells with activated RAS resulted in radiosensitization, whereas treatment of cells with wild-type RAS had no effect on radiation survival. Here we ask whether the findings obtained in vitro have applicability in vivo. We found that treatment of nude mice bearing T24 tumor cell xenografts with farnesyltransferase inhibitors resulted in a significant and synergistic reduction in tumor cell survival after irradiation. The regrowth of T24 tumors expressing activated RAS was also significantly prolonged by the addition of treatment with farnesyltransferase inhibitors compared to the regrowth after irradiation alone. In contrast, there was no effect on the radiosensitivity of HT-29 tumors expressing wild-type RAS. These results demonstrate that specific radiosensitization of tumors expressing activated RAS oncogenes can be obtained in vivo.

Alkyl and Aryl Transferases↗
PubMed1999

p34(Cdc2) kinase activity is excluded from the nucleus during the radiation-induced G(2) arrest in HeLa cells.

The progression of cells from G(2) into mitosis is blocked by exposure to DNA-damaging agents such as ionizing radiation. This G(2) delay is associated with reduced cyclin B1-specific associated histone H1 kinase activity, increased inhibitory phosphorylation of p34(Cdc2), and depressed cyclin B1 levels in HeLa cells. Induction of cyclin B1 or expression of Cdc2AF, a mutant p34(Cdc2) that lacks the sites of inhibitory phosphorylation, only partially reverses the radiation-associated G(2) delay, although both maneuvers rapidly result in increased histone H1 kinase activity. To account for the persistent G(2) delay in the face of active p34(Cdc2) kinase, we determined the location of the kinase activity. Although p34(Cdc2) was active in the cytoplasm, the nuclear p34(Cdc2) was inactive. Irradiation led to nuclear accumulation of the inactive tyrosine-phosphorylated form of p34(Cdc2), whereas the active form was seen in the cytoplasm. At later times when cells had resumed cell cycle progression, nuclear kinase activity was detectable. These results give evidence of segregation of cytoplasmic and nuclear kinase activity after DNA damage that has the effect of enhancing checkpoint control. Shielding the nucleus from the potentially deleterious effects of kinase activity after DNA damage may help irradiated human cancer cells respond to irradiation.

CDC2 Protein Kinase↗
PubMed1999

'Proteolytic switching': opposite patterns of regulation of gelatinase B and its inhibitor TIMP-1 during human melanoma progression and consequences of gelatinase B overexpression.

Although it is generally accepted that proteolytic degradation is an important mechanism used by malignant cells in the process of metastasis, comparatively little is known about the regulation of molecules responsible for proteolysis and how they become de-regulated during human tumour progression. Using a genetically related pair of human melanoma cell lines, derived from the same patient at different stages of disease, we analysed differences in the cytokine-mediated regulation of gelatinase B (MMP-9), an enzyme thought to play an important role in cellular invasiveness, and TIMP-1, a physiological inhibitor of this enzyme. Whereas the advanced stage (i.e. metastatic) partner of this pair (WM 239) could produce gelatinase B upon induction with interleukin (IL)-1beta or tumour necrosis factor alpha (TNF-alpha), the early stage (i.e. primary) partner (WM 115) could not. In sharp contrast, we found that TIMP-1 displayed an opposite pattern of induction in these cell lines. Specifically, the early stage cell line, WM 115, demonstrated a marked increase in the production of TIMP-1 when treated with IL-1beta or TNF-alpha whereas the advanced cell line, WM 239, showed no such increase. Treatment with the DNA demethylating agent, 2-deoxy-5-azacytidine, resulted in a marked up-regulation of both gelatinase B and TIMP-1 in both cell lines. It was further found that constitutive overexpression of gelatinase B in WM 239 cells and an additional melanoma cell line (MeWo), derived from a metastatic lesion, was able to greatly enhance lung colonization in an experimental metastasis assay while we did not observe differences in tumorigenicity. From these results we conclude that an altered responsiveness of gelatinase B and TIMP-1 to induction by similar agents is associated with disease progression in human melanoma and that this altered responsiveness can have consequences to the aggressive nature of the disease.

Animals↗
PubMed1999

Therapeutic resistance: characterization and inactivation by specific antiserum of a putative protein family produced by tumour cells.

A multitherapy resistance (MTR) factor produced by Cloudman S91 mouse melanoma cells rescues a responsive cell line after gamma-irradiation, short wavelength ultraviolet light, mitomycin C, vinblastine and actinomycin D. A similar activity with respect to ionizing radiation is now shown to be produced by human melanoma cells and by both human and mouse breast cancer cells but not by five normal cell lines. In these studies, the factor produced in serum-free conditioned medium (SFCM) by Cloudman S91/I3 cells is further characterized. Its activity in a clonogenic assay using related Cloudman S91/amel cells is destroyed by trypsin but not by DNase and is stable for at least 8 days at a variety of temperatures including 37 degrees C. Molecules greater than 30 kDa from SFCM collected from S91/I3 cells were concentrated and separated by preparative zonal electrophoresis (PZE). Bioactivity was present in both the cathode- and the anode-running fractions. The active acidic (anode) fractions were analysed by preparative isoelectric focusing. Bioactivity was present between pI 3.5 and 4.2. These PZE fractions were also used to immunize two rabbits, both of which produced antiserum that abrogated the bioactivity of SFCM and of the PZE cathode fractions. Antiserum also decreased the survival of irradiated S91/I3 producer cells that do not respond to SFCM but nonetheless must require MTR proteins for the expression of radiation resistance. These studies present a model for the production of rescue factors by non-clonogenic tumour cells that may persist in some tumours for considerable periods of time.

Amino Acid Transport Systems, Neutral↗