An antisense oligonucleotide targeted to human Ku86 messenger RNA sensitizes M059K malignant glioma cells to ionizing radiation,bleomycin, and etoposide but not DNA cross-linking agents

Ku is a heterodimer of M(r) 70,000 and M(r) 86,000 subunits. It binds with strong affinity to DNA ends and is indispensable for nonhomologous DNA end joining (NHEJ) and V(D)J recombination. In this study, we investigated whether down-regulation of the Ku86 gene, by 2'-O-methoxyethyl/uniform phosphorothioate chimeric antisense oligonucleotides (ASOs), increases the sensitivity of the DNA-protein kinase catalytic subunit (PKcs)-proficient human glioma cell line (M059K), and its isogenic DNA-PKcs-deficient counterpart (M059J), to ionizing radiation and anticancer drugs. Transfection of these cell lines with 200 nM Ku86 antisense ASOs was associated with a specific decrease in Ku86 mRNA levels (IC(50) <25 nM; n = 3) and a concomitant rapid decrease (<10% of control) in Ku86 protein expression. Moreover, transfection of M059K cells with Ku86 antisense ASOs markedly increased cell death after treatment with ionizing radiation, bleomycin, and etoposide. However, no sensitization to the DNA cross-linking agents chlorambucil and cisplatin was observed after Ku86 antisense transfection. As expected, transfection of M059J cells with Ku86 antisense ASOs did not result in any sensitization to ionizing radiation, bleomycin, or DNA cross-linking agents, but there was a 2-fold increase in sensitivity to etoposide. Thus, our results indicate that antisense ASOs targeted against Ku86 may increase the efficacy of radiotherapy and DNA-damaging agents in tumor treatment. Furthermore, Ku86 antisense ASOs may be used to create a temporal knockout in different human cell lines to further investigate the biological roles of Ku86.     

Influence of oxygen on the radiosensitivity of human glioma cell lines

We have investigated the influence of hypoxia on the radiosensitivity of 4 early-passage tumor cell lines that were established from malignant glioma patients at our Institute. These cell lines were M006, M059J (a highly radiosensitive line), M059K (a radioresistant line derived from the same biopsy as M059J), and M010b. The GM637 human fibroblast cell line was used as a normal control. The oxygen enhancement ratios (OERs) for these cell lines, determined using a clonogenic survival assay, were approximately 3.6 (GM637), approximately 3.7 (M006), approximately 2.5 (M010b), approximately 2.1 (M059K), and approximately 3.5 (M059J). The broad range of OERs for these glioma lines was not related to cellular glutathione levels or to differences in intrinsic cellular radiosensitivity. Because studies with rodent cell lines indicate that defects in certain DNA repair genes, including ERCC1, can greatly influence cellular OERs, and because several such repair genes, including ERCC1, localize to a region of chromosome 19q that is close to a common deletion in human glioma, we reasoned that such deletions might contribute to the diverse OERs of these tumor cell lines. However, measurements of ERCC1 protein levels using immunofluorescence staining or Western blotting, of ERCC1 mRNA levels using Northern blotting, and of functional nucleotide excision repair capability using the UV/adenovirus reactivation assay, failed to indicate any deficit in these activities. Thus, although the effect of hypoxia on the radiosensitivity of different human glioma cell lines can vary widely, the mechanism of this effect remains unknown. The potential implications of this finding for radiation therapy, and especially for hypoxia imaging-guided intensity-modulated radiation therapy (IMRT) treatment planning, are discussed.     

Lovastatin sensitized human glioblastoma cells to TRAIL-induced apoptosis

Synergy study with chemotherapeutic agents is a common in vitro strategy in the search for effective cancer therapy. For non-chemotherapeutic agents, efficacious synergistic effects are uncommon. Here, we have examined two non-chemotherapeutic agents for synergistic effects: lovastatin and Tumor Necrosis Factor (TNF)-related apoptosis-inducing ligand (TRAIL) for synergistic effects; on three human malignant glioblastoma cell lines, M059K, M59J, and A172. Cells treated with lovastatin plus TRAIL for 48 h showed 50% apoptotic cell death, whereas TRAIL alone (1,000 ng/ml) did not, suggesting that lovastatin sensitized the glioblastoma cells to TRAIL attack. Cell cycle analysis indicated that lovastatin increased G₀–G₁ arrest in these cells. Annexin V study demonstrated that apoptosis was the predominant mode of cell death. We conclude that the combination of lovastatin and TRAIL enhances apoptosis synergistically. Moreover, lovastatin sensitized glioblastoma cells to TRAIL, suggesting a new strategy to treat glioblastoma.     

Overexpression of cyclooxygenase-2 is associated with radioresistance in oral squamous cell carcinoma

Cyclooxygenase-2 (COX-2), an inducible isoform of cyclooxygenase, is overexpressed in many types of malignant tumors, which in turn may stimulate tumor growth and protect against damage by irradiation or cytotoxic agents. The purpose of this study is to investigate the relationship between the radiation sensitivity and elevated level of COX-2. Radiation sensitivity of the eight oral SCC cell lines differed greatly in their response to radiation. Further, the level of the COX-2 expression correlated inversely with increased tumor radiation sensitivity. The similar significant association between the response to preoperative radiation therapy and COX-2 overexpression was observed in the oral SCC patients. In addition, treatment with a COX-2 selective inhibitor enhanced the radioresponse of HSC-2 cell, which constitutively expressed COX-2. These results suggested that COX-2 expression level correlates to radiation tolerance and the COX-2 selective inhibitor may be a potent enhancer for tumor radioresponse in oral SCC.     

Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes malignant glioma cells by inducing autophagy

DNA-dependent protein kinase (DNA-PK) plays a major role in the repair of DNA double-strand breaks induced by ionizing radiation (IR). Lack of DNA-PK causes defective DNA double-strand break repair and radiosensitization. In general, the cell death induced by IR is considered to be apoptotic. On the other hand, nonapoptotic cell death, autophagy, has recently attracted attention as a novel response of cancer cells to chemotherapy and IR. Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm. Little is known, however, regarding the relationship between DNA-PK and IR-induced autophagy. In the present study, we used human malignant glioma M059J and M059K cells to investigate the role of DNA-PK in IR-induced apoptotic and autophagic cell death. Low-dose IR induced massive autophagic cell death in M059J cells that lack the catalytic subunit of DNA-PK (DNA-PKcs). Most M059K cells, the counterpart of M059J cells in which DNA-PKcs are expressed at normal levels, survived, and proliferated although a small portion of the cells underwent apoptosis. Low-dose IR inhibited the phosphorylation of p70(S6K), a molecule downstream of the mammalian target of rapamycin associated with autophagy in M059J cells but not in M059K cells. The treatment of M059K cells with antisense oligonucleotides against DNA-PKcs caused radiation-induced autophagy and radiosensitized the cells. Furthermore, antisense oligonucleotides against DNA-PKcs radiosensitized other malignant glioma cell lines with DNA-PK activity, U373-MG and T98G, by inducing autophagy. The specific inhibition of DNA-PKcs may be promising as a new therapy to radiosensitize malignant glioma cells by inducing autophagy.     

Different G2/M accumulation in M059J and M059K cells after exposure to DNA double-strand break-inducing agents

PURPOSE: To investigate and compare the cell cycle progression in relation to cell death in the human glioma cell lines, M059J and M059K, after exposure to DNA double-strand break-inducing agents. METHODS AND MATERIALS: The M059J and M059K cells, deficient and proficient in the catalytic subunit of the DNA-dependent protein kinase, respectively, were exposed to 1 and 4 Gy of photons or accelerated nitrogen ions. In addition, M059J and M059K cells were treated with 10 and 40 mug/mL of bleomycin for 30 min, respectively. Cell cycle progression, monitored by DNA flow cytometry, was measured up to 72 h after treatment. RESULTS: M059J, but not M059K, cells displayed G(2)/M accumulation after low linear energy transfer irradiation. High linear energy transfer radiation exposure however, resulted in a substantial increase of M059K cells in the G(2)/M phase detected at 48 h. At 72 h, the number of cells in the G(2)/M phase was equivalent to its control. M059J cells accumulated mainly in S phase after high linear energy transfer irradiation. In contrast to M059K, M059J cells were still blocked at 72 h. Bleomycin induced G(2)/M accumulation for both M059J and M059K cells detected 24 h after treatment. At 48 h, the percentage of bleomycin-treated M059J cells in G(2)/M phase remained high, and the number of M059K cells had decreased to control levels. Neither cell line showed cell cycle arrest (< or =10 h) after exposure to these agents. CONCLUSION: Distinct cell cycle block and release is dependent on the complexity of the induced DNA damage and the presence of the DNA-dependent protein kinase catalytic subunit.     

Sensitivity of human melanoma cells to L-dopa and DL-buthionine (S,R)-sulfoximine

Four of seven human melanoma cell lines were sensitive to killing by L-dopa (D37 1.0-4.7 microM) compared with fibroblasts, Hela, and three ovarian tumor cell lines (D37 12-59 microM). All seven melanoma lines, however, were sensitive to DL-buthionine(S,R)sulfoximine (BSO) (D37 0.73-8.5 microM) compared with the nonmelanoma cells (D37 25-68 microM). The melanoma line most sensitive to BSO (MM418) was highly melanized, proliferated slowly and was resistant to other agents [dopa, 5-(3-methyl-1-triazeno)5-imidazole-4-carboxamide, melphalan, methotrexate, hydroxyurea, etoposide, Adriamycin]. In most cell lines, L-dopa and BSO blocked cell proliferation in all phases of the cell cycle. Cellular sensitivity to dopa or BSO did not correlate with levels of total soluble SH, glutathione (GSH), GSH reductase, GSH peroxidase or GSH transferase, or with the extent of GSH depletion induced by the drug. No GSH transferase activity could be detected in the dopa-resistant HeLa line, indicating that detoxification of quinones is not an important mechanism of resistance. Within the group of melanoma cell lines, sensitivity to dopa correlated with decreased level of gamma-glutamyl transpeptidase (r = 0.81). However, the gamma-glutamyl transpeptidase inhibitor azaserine was less effective than BSO in enhancing the toxicity of dopa. It can be inferred that (a) there is no simple relationship between GSH metabolism and sensitivity to dopa or BSO in human melanoma cells, and (b) BSO may be an effective agent for melanoma.     

CD95/CD95 ligand-independent potentiation of treosulfan cytotoxicity by BSO in malignant glioma cells in vitro and in vivo

The glutathione synthase inhibitor, buthionine sulfoximine (BSO), specifically enhances the cytotoxic effects of treosulfan in human glioma cells. BSO depletes glutathione and greatly enhances treosulfan cytotoxicity in all the 12 human malignant glioma cell lines examined. None of these cell lines showed enhanced susceptibility to CD95L- or tumor necrosis factor (TNF)-alpha-induced apoptosis when glutathione is depleted. The combination of serial systemic BSO applications (300 mg/kg) and a single systemic dose of treosulfan (2.5 g/kg) reduced the growth of intracranially growing rat C6 gliomas in vivo by 73% whereas treosulfan alone reduced tumor growth by 16% and BSO alone had no effect. BSO lowered glutathione levels to 25-30% in peripheral blood mononuclear cells (PBMC) and to 50% in the glioma tissue. The glutathione levels in the non-tumor-bearing contralateral hemisphere were unaffected by systemic BSO treatment. The main side effects of treosulfan, gastrointestinal and bone marrow toxicity, were not significantly enhanced by BSO.     

Effects of glutathione depletion by buthionine sulfoximine on the sensitivity of EMT6/SF cells to chemotherapy agents or X radiation

The effects of depletion of cellular glutathione (GSH) on the sensitivity of cultured EMT6/SF cells to chemotherapy agents or x rays under hypoxic and aerated conditions were investigated. Buthionine sulfoximine (BSO), a potent inhibitor of the enzyme gamma-glutamyl-cysteine synthetase, was used to deplete cellular GSH. Addition of BSO (50 microM) to EMT6/SF cultures depleted cellular GSH with a half-time of approximately 2 hr. Cellular GSH reached very low levels within hours of addition of BSO. After removal of BSO, cellular GSH recovered with approximately the same kinetics as was seen for depletion. Incubation of EMT6/SF cells with BSO concentrations of up to 1 mM did not reduce the viability or inhibit growth when exposure was limited to times less than 24 hr. However, for longer exposure times, toxicity and growth inhibition were demonstrated in a dose dependent fashion. EMT6/SF cells were treated with chemotherapy agents under either aerated or extremely hypoxic conditions. Cells were more sensitive to cis-dichlorodiammino Pt(II) (DDP), mitomycin C (MitC), L-phenylalanine mustard (L-PAM), and nitrogen mustard (HN2) when treatment was under hypoxic conditions. The magnitude of this sensitization under hypoxic conditions ranged from a dose modifying factor (DMF) of 1.4 (HN2) to 4.1 (MitC), measured at the 0.1 level of cell survival. Hypoxic EMT6/SF cells were more resistant to the cytotoxic effects of actinomycin D (ActD) under hypoxic conditions (DMF = 10 at SF = 0.3). When cellular GSH was depleted to less than 5% of control by treatment with 50 microM BSO for 12-14 hr, cells were sensitized to DDP, L-PAM and HN2 under both aerated and hypoxic conditions. DMF's ranged from 1.4-6.5, depending on the agent. Hypoxic cell sensitization was never significantly greater than that seen in aerated cells, as was the case for X radiation (DMF = 1.3 for hypoxic cells only). GSH depletion also sensitized to MitC, but only under aerated conditions (DMF = 2.1). Hypoxic EMT6/SF cells were not sensitized to MitC by depletion of GSH. GSH depletion afforded slight protection against ActD toxicity under both aerated and hypoxic conditions. These studies suggest that cellular GSH plays an important role in modifying cellular response to cytotoxic drugs. GSH depletion may sensitize tumor cells to some chemotherapy agents, but differential sensitization of tumors compared to normal tissues, based on hypoxic tumor cells as targets, would not be expected based on these in vitro experiments.     

BSO-induced reduction of glutathione levels increases the cellular radiosensitivity of drug-resistant human tumor cells.

Acquired resistance to cis-platinum and melphalan, in the human ovarian OAW42 tumor cell line, respectively, conferred a 3- and 1.5-fold decrease in photon sensitivity. Analysis of cell survival curves by the linear quadratic equation showed an accompanying 5- and 2-fold reduction in the magnitude of the initial slope (alpha). Treatment with the GSH depleting agent BSO restored the magnitude of alpha to a value similar to that of the parental line without evidence of dose modification in the high-dose region of the cell survival curve. This in conjunction with failure of alteration in GSH levels to affect parental OAW2 sensitivity and of the SER of BSO to reflect GSH levels suggest a possible GSH independent mechanism of action for BSO. If similar patterns occur in the clinic, the possibility exists of circumventing collateral resistance between chemotherapeutic agents and ionizing radiation, provided that tumor thiol levels can be preferentially depleted.     

Response of xenografts of human malignant gliomas and squamous cell carcinomas to fractionated irradiation.

The response of xenografts of five human malignant glioma cell lines and two human squamous cell carcinomas to fractionated irradiation was studied. For this, the tumors were transplanted into nude mice which had been further immunosuppressed by 6 Gy whole-body irradiation. Radiation was given as 30 fractions applied under normal blood flow conditions in two sessions per day over 15 days. Absolute and specific tumor growth delay after 48 Gy, and tumor control dose 50% (TCD50) were evaluated. Using local tumor control as experimental endpoint, four out of five malignant gliomas were more resistant to fractionated radiation therapy than the two squamous cell carcinomas. The TCD50s of these four gliomas ranged from 73 Gy to more than 120 Gy, whereas the TCD50s of the squamous cell carcinomas were 51 and 60 Gy. Absolute tumor growth delay correlated well with TCD50, but no correlation was obtained between specific growth delay and TCD50. The response of the human tumor xenografts in vivo did not correlate with the surviving fractions at 2 Gy of the same cell lines in vitro which have been previously obtained in our laboratory. The results suggest that the unique radioresistance observed in malignant gliomas in patients is at least in part reflected in human tumor xenografts. The lack of correlation between the surviving fraction at 2 Gy in vitro and the tumor response in vivo could be a consequence of an immune response by the host, a difference in cell radiation sensitivity between cell lines and xenografted tumors, or of differences of parameters such as hypoxic fraction, rate of repopulation, and cell cycle effects between the different tumor lines studied. It illustrates the difficulties which might be involved in the prediction of the response of individual tumors to radiation therapy based solely on the intrinsic radiosensitivity of the tumor cells as assayed by in vitro assays of colony formation.     

Glutathione as a determinant of cellular response to doxorubicin

We have studied in detail the relationship between glutathione (GSH) depletion and sensitivity of HEp3 human carcinoma cells to doxorubicin [Adriamycin (ADR)]. Exponentially growing HEp3 cells were incubated with L-buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, for different periods so that a range of GSH depletion could be obtained. These GSH-depleted cells were then treated with a combination of BSO and ADR (1 microgram/ml) for various durations. Under these conditions, the cytotoxicity of ADR was significantly enhanced by GSH depletion. The extent of ADR dose enhancement was found to be inversely proportional to cellular GSH level at the time of ADR treatment. Furthermore, it was shown that the dose-enhancement factors (DEF) also correlated with the duration of combined BSO and ADR treatment. For example, at a GSH level of 45% of untreated control, 18.5 +/- 3 fmol/cell or 4.8 +/- 0.3 X 10(-3) fmol/mum3 (+/- SD), DEF of 8.0, 6.4, and 5.0 were obtained for treatment periods of 3 hours, 2 hours, and 1 hour, respectively. Further study showed that the GSH kinetics differed significantly for the different treatment times, which indicates that GSH kinetics may be an important factor in determining the intrinsic sensitivity of HEp3 cells to ADR. Furthermore, the kinetics of GSH response to ADR varied significantly between cell lines. In the study of the effect of such differences, the GSH kinetics of 3 human ovarian tumor cell lines with different intrinsic sensitivities to ADR were investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison between the in vitro intrinsic radiation sensitivity of human soft tissue sarcoma and breast cancer cell lines

The purpose of this study is to evaluate the radiation sensitivity of human soft tissue sarcoma cell lines in vitro and to compare with that of human breast carcinoma and glioblastoma cell lines. The intrinsic radiation sensitivity parameters of seven human soft tissue sarcomas and eight breast carcinoma cell lines were investigated in vitro by clonogenic assays for single-dose irradiation under aerobic conditions on cells in exponential phase of growth. The results for sarcoma cell lines showed that the mean surviving fraction at 2 Gy (SF2) was 0.39 (SD ± 0.09) with a range of 0.24 to 0.53, and the average mean inactivation dose (MID) was 1.92 (SD ± 0.35) range from 1.36 Gy to 2.49 Gy. These values were not different from that of breast cell lines examined concurrently and using the same experimental methods (mean SF2 0.38, SD ± 0.09; MID 1.9 Gy, SD ± 0.37). However, radiobiological parameters of nine karyotyped human malignant glioma cell lines determined earlier in this laboratory were significantly higher (mean SF2 0.50 ± 0.14; mean MID 2.61 ± 0.60). In conclusion, the data presented here do not support the view that cells of sarcomas show unusual radiation resistance. To the extent that the in vitro determined cellular radiation sensitivity reflects the tumor response in vivo, the success rate for radiation applied against sarcoma and breast carcinoma of comparable size could be similar. © 1996 Wiley-Liss, Inc.     

X-ray and cis-diamminedichloroplatinum(II) cross-resistance in human tumor cell lines

Clinical studies have suggested a close correlation between cis-diamminedichloroplatinum(II) (cisplatin) and radiation resistance. To determine whether this cross-resistance is due to an inherent cellular resistance to both agents, ten early passage human tumor cell lines were examined for their radiation and cisplatin sensitivity in vitro. Previous studies have suggested that these early passage tumor cell lines retain many of their in vivo characteristics and are therefore good models for tumor cells in vivo. Radioresistance was strongly associated with cisplatin resistance in these cell lines. Four of the cell lines examined were radioresistant, having Dos greater than 2.0 Gy. These four lines were also resistant to cisplatin, with the dose reducing survival to 10% greater than 1.29 microM. The remaining six cell lines had Dos ranging from 1.07 to 1.57 Gy of X-ray and doses reducing survival to 10% of less than 0.83 microM cisplatin. Because early passage human tumor cell lines were used, resistance or sensitivity to radiation and cisplatin most likely developed in vivo and was not due to selection in vitro. These results indicate that cross-resistance between cisplatin and radiation in vivo is probably due primarily to an inherent cellular resistance to these agents and not necessarily to the tumor microenvironment in situ.     

Sensitization of human carcinoma cells to alkylating agents by small interfering RNA suppression of 3-alkyladenine-DNA glycosylase

One of the major cytotoxic lesions generated by alkylating agents is DNA 3-alkyladenine, which can be excised by 3-alkyladenine DNA glycosylase (AAG). Inhibition of AAG may therefore result in increased cellular sensitivity to chemotherapeutic alkylating agents. To investigate this possibility, we have examined the role of AAG in protecting human tumor cells against such agents. Plasmids that express small interfering RNAs targeted to two different regions of AAG mRNA were transfected into HeLa cervical carcinoma cells and A2780-SCA ovarian carcinoma cells. Stable derivatives of both cell types with low AAG protein levels were sensitized to alkylating agents. Two HeLa cell lines with AAG protein levels reduced by at least 80% to 90% displayed a 5- to 10-fold increase in sensitivity to methyl methanesulfonate, N-methyl-N-nitrosourea, and the chemotherapeutic drugs temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea. These cells showed no increase in sensitivity to UV light or ionizing radiation. After treatment with methyl methanesulfonate, AAG knockdown HeLa cells were delayed in S phase but accumulated in G2-M. Our data support the hypothesis that ablation of AAG activity in human tumor cells may provide a useful strategy to enhance the efficacy of current chemotherapeutic regimens that include alkylating agents.     

Evaluation of nitroimidazole hypoxic cell radiosensitizers in a human tumor cell line high in intracellular glutathione

Five nitroimidazole hypoxic cell radiosensitizers were evaluated in a human lung adenocarcinoma cell line (A549) whose GSH level was 8-fold higher than Chinese hamster V79 cells. One millimolar concentrations of Misonidazole (MISO), SR-2508, RSU-1164, RSU-1172, and Ro-03-8799 sensitized hypoxic A549 cells to radiation, with Ro-03-8799 giving the highest sensitizer enhancement ration (SER) (2.3). However, MISO, SR-2508 and Ro-03-8799 were less effective in this cell line than in V79 cells, presumably due to higher GSH content of the A549 cells. Increased hypoxic radiosensitization was seen with 0.1 mM Ro-03-8799 after GSH depletion by BSO as compared to 0.1 mM Ro-03-8799 alone (SER-1.8 vs 1.3). The combination of GSH depletion and 0.1 mM Ro-03-8799 was considerably more toxic than 0.1 mM or 1.0 mM Ro-03-8799 alone. This sensitivity was much greater than has been observed for SR-2508. These data show that Ro-03-8799 was the most efficient hypoxic cell radiosensitizer in a human tumor cell line considerably higher in GSH than the rodent cell lines often used in hypoxic radiosensitization studies. Thus, Ro-03-8799 may be a more effective hypoxic cell sensitizer in human tumors that are high in GSH.     

Flavopiridol potentiates the cytotoxic effects of radiation in radioresistant tumor cells in which p53 is mutated or Bcl-2 is overexpressed

PURPOSE: Loss of the cell-cycle regulatory protein p53 or overexpression of the antiapoptotic protein Bcl-2 is associated with resistance to radiation in several types of cancer cells. Flavopiridol, a synthetic flavone, inhibits the growth of malignant tumors cells in vitro and in vivo through multiple mechanisms. The purpose of the present study is to clarify whether flavopiridol enhances the cytotoxic effects of radiation in tumor cells that contain dysfunction p53 or that overexpress Bcl-2. METHODS AND MATERIALS: A human glioma cell line (A172/mp53) stably transfected with a plasmid containing mutated p53 and a human cervical cancer cell line (HeLa/bcl-2) transfected with a bcl-2 expression plasmid were used. Cells were incubated with flavopiridol for 24 h after radiation, and then cell viability was determined by a colony formation assay. Foci of phosphorylated histone H2AX were also evaluated as a sensitive indicator of DNA double-strand breaks. RESULTS: Compared with the parental wild-type cells, both transfected cell lines were more resistant to radiation. Post-treatment with flavopiridol increased the cytotoxic effects of radiation in both transfected cell lines, but not in their parental wild-type cell lines. Post-treatment with flavopiridol inhibited sublethal damage repair as well as the repair of DNA double-strand breaks in response to radiation. CONCLUSIONS: Flavopiridol enhanced the cytotoxic effect of radiation in radioresistant tumor cells that harbor p53 dysfunction or Bcl-2 overexpression. A combination treatment of flavopiridol with radiation has the potential to conquer the radioresistance of malignant tumors induced by the genetic alteration of p53 or bcl-2.     

Current results and perspectives of the use of human tumor cell lines for antitumor drug screening

Cytotoxic screening of 2,000 chemical compounds against human tumor cell lines has identified certain antimetabolities, platinum complexes, actinomycin D derivatives and somatostatin analogs as possible antitumor agents. Differential sensitivity of such lines derived from five cancers (lung, colon, ovary, breast and leukemia) to certain antitumor agents has been established. This panel of cell lines is currently employed as a model for in vitro drug screening to identify biological response profile at earlier stages.     

Effect of BSO on the radiation response at low (0-4 Gy) doses.

Depletion of thiols by various agents has been shown to radiosensitize hypoxic cells. The agent BSO is used to specifically inhibit GSH synthesis, and the effect of this treatment on radiation response, particularly in the region of clinical interest, was studied using automated microscopic identification of cells for measurement of high survival levels. In both CHO and V79 cell lines, the enhancement rations were greater at low doses (80% S) than at high doses (2% S) in cells irradiated in hypoxia. GSH depletion also affected the aerobic response in both dose regions, with again higher ER's observed at low doses. These results support previous studies which suggest that GSH levels affect the shoulder portion of the survival curve. However, as with untreated cells, the OER's remain higher at high doses than at low doses, in BSO treated cells of both lines. These studies complement an earlier low-dose study on diamide by Skarsgard and co-workers, and were carried out to increase our understanding of the factors which affect radiation sensitivity in the range of doses used in fractionated regimens in the clinic.