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Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin H synthase, has been implicated in the growth and progression of a variety of human cancers. Although COX-2 overexpression has been observed in human gliomas, the prognostic or clinical relevance of this overexpression has not been investigated to date. In addition, no study has analyzed the relationship between COX-2 expression and other molecular alterations in gliomas. Consequently, we examined COX-2 expression by immunohistochemistry in tumor specimens from 66 patients with low- and high-grade astrocytomas and correlated the percentage of COX-2 expression with patient survival. We also analyzed the relative importance of COX-2 expression in comparison with other clinicopathological features (age and tumor grade) and other molecular alterations commonly found in gliomas (high MIB-1 level, p53 alteration, loss of retinoblastoma (Rb) protein or p16, and high bcl-2 level). Kaplan-Meier analyses demonstrated that high COX-2 expression (>50% of cells stained positive) correlated with poor survival for the study group as a whole (P < 0.0001) and for those with glioblastoma multiforme in particular (P < 0.03). Cox regression analyses demonstrated that COX-2 expression was the strongest predictor of outcome, independent of all other variables. In addition, high COX-2 expression correlated with increasing histological grade but did not correlate with positive p53 immunostaining, bcl-2 expression, loss of p16 or retinoblastoma protein expression, or high MIB-1 expression. These findings indicate that high COX-2 expression in tumor cells is associated with clinically more aggressive gliomas and is a strong predictor of poor survival.

The antitumor effects of the selective cyclooxygenase (COX)-2 inhibitor SC-236 alone and in combination with radiation were investigated using the human glioma cell line U251 grown in monolayer culture and as tumor xenografts. On the basis of Western and Northern blot analyses, these cells express COX-2 protein and mRNA to levels similar to those in the human colon carcinoma cell line HT29. Treatment of U251 cells in monolayer culture with 50 microM SC-236 resulted in a time-dependent decrease in cell survival as determined by a clonogenic assay. The cell death induced by SC-236 was associated with apoptosis and the detachment of cells from the monolayer. After 2 days of drug treatment, the cells that remained attached were exposed to graded doses of radiation, and the clonogenic assay was performed. Comparison of the survival curves for drug-treated and untreated cultures revealed that SC-236 enhanced radiation-induced cell death. In these combination studies, SC-236 treatment resulted in a dose-enhancement factor of 1.4 at a surviving fraction of 0.1, with the surviving fraction at 2 Gy (SF2) reduced from 0.61 to 0.31. These data indicate that in vitro SC-236 induces U251 apoptotic cell death and enhances the radiosensitivity of the surviving cells. To extend these investigations to an in vivo situation, U251 glioma cells were grown as tumor xenografts in the hind leg of nude mice, and SC-236 was administered in drinking water. SC-236 alone slowed tumor growth rate, and when administered in combination with local irradiation, SC-236 caused a greater than additive increase in tumor growth delay. These in vitro and in vivo results suggest that the selective inhibition of COX-2 combined with radiation has potential as a cancer treatment.

Objective: The rationale and current evidence for using p53 gene replacement as a potential treatment for human gliomas are reviewed. The possible benefits of and obstacles to this approach are delineated., Methods: One approach to overcoming the poor outcomes associated with conventional glioma therapies involves the replacement of tumor suppressor genes that are fundamental to glioma development. The p53 gene is one of the most frequently mutated genes in human gliomas, and loss of p53 function is critical to the development of glial neoplasms. Consequently, replacement of the p53 gene using viral vectors may be a potential treatment for human gliomas., Results: In vitro studies demonstrate that adenovirus-mediated p53 gene transfer into gliomas with mutant p53 results in massive apoptosis. Similarly, transfer of p53 inhibits tumor growth in vivo. In contrast to mutant p53 gliomas, wild-type p53 glioma cells are resistant to the apoptotic effects of p53 transfer, but this resistance can be overcome by the addition of deoxyribonucleic acid-damaging agents such as ionizing radiation or chemotherapy. The main obstacle to p53 gene therapy involves the limitations associated with current modes of delivery., Conclusion: Preclinical data strongly support the use of p53 gene transfer as a potential treatment for human gliomas.

Object: The authors sought to determine whether combining p53 gene transfer with radiation therapy would enhance the therapeutic killing of p53 wild-type glioma cells. It has been shown in several reports that adenovirus-mediated delivery of the p53 gene into p53 mutant gliomas results in dramatic apoptosis, but has little effect on gliomas containing wild-type p53 alleles. Therefore, p53 gene therapy alone may not be a clinically effective treatment for gliomas because most gliomas are composed of both p53 mutant and wild-type cell populations. One potential approach to overcome this problem is to exploit the role p53 plays as an important determinant in the cellular response to ionizing radiation., Methods: In vitro experiments were performed using the glioma cell line U87MG, which contains wild-type p53. Comparisons were made to the glioma cell line U251MG, which contains a mutant p53 allele. Monolayer cultures were infected with an adenovirus containing wild-type p53 (Ad5CMV-p53), a control vector (dl312), or Dulbecco's modified Eagle's medium (DMEM). Two days later, cultures were irradiated and colony-forming efficiency was determined. Transfection with p53 had only a minor effect on the plating efficiency of nonirradiated U87MG cells, reducing the plating efficiency from 0.23 +/- 0.01 in DMEM to 0.22 +/- 0.04 after addition of Ad5CMV-p53. However, p53 transfection significantly enhanced the radiosensitivity of these cells. The dose enhancement factor at a surviving fraction of 0.10 was 1.5, and the surviving fraction at 2 Gy was reduced from 0.61 in untransfected controls to 0.38 in p53-transfected cells. Transfection of the viral vector control (dl312) had no effect on U87MG radiosensitivity. In comparison, transfection of Ad5CMV-p53 into the p53 mutant cell line U251 MG resulted in a significant decrease in the surviving fraction of these cells compared with controls, and no radiosensitization was detected. To determine whether Ad5CMV-p53-mediated radiosensitization of U87MG cells involved an increase in the propensity of these cells to undergo apoptosis, flow cytometric analysis of terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridinetriphosphate nick-end labeling-stained cells was performed. Whereas the amount of radiation-induced apoptosis in uninfected and dl312-infected control cells was relatively small (2.1 +/- 0.05% and 3.7 +/- 0.5%, respectively), the combination of Ad5CMV-p53 infection and radiation treatment significantly increased the apoptotic frequency (18.6 +/- 1.4%). To determine whether infection with Ad5CMV-p53 resulted in increased expression of functional exogenous p53 protein, Western blot analysis of p53 was performed on U87MG cells that were exposed to 9 Gy of radiation 2 days after exposure to Ad5CMV-p53, dl312, or DMEM. Infection with Ad5CMV-p53 alone increased p53 levels compared with DMEM- or dl312-treated cells. Irradiation of AdSCMV-p53-infected cells resulted in a further increase in p53 that reached a maximum at 2 hours postirradiation. To determine whether exogenous p53 provided by Ad5CMV-p53 had transactivating activity, U87MG cells were treated as described earlier and p21 messenger RNA levels were determined. Infection of U87MG cells with Ad5CMV-p53 only resulted in an increase in p21 compared with DMEM- and dl312-treated cells. Irradiation of AdSCMV-p53-infected cells resulted in an additional time-dependent increase in p21 expression., Conclusions: These data indicate that adenovirus-mediated delivery of p53 may enhance the radioresponse of brain tumor cells containing wild-type p53 and that this radiosensitization may involve converting from a clonogenic to the more sensitive apoptotic form of cell death. Although the mechanism underlying this enhanced apoptotic susceptibility is unknown, the AdSCMV-p53-infected cells have a higher level of p53 protein, which increases further after irradiation, and this exogenous p53 is transcriptionally active. (ABSTRACT TRUNCATE

In an earlier study we showed that there is a good correlation between sister chromatid exchange induction and cell kill in 9L cells treated with certain nitrosoureas. In the study reported here, we treated four 9L cell lines that have different sensitivities to chloroethylnitrosoureas with 1,3-bis (2-chloroethyl)-1-nitrosourea, chlorozotocin, and ethylnitrosourea and determined the number of sister chromatid exchanges induced. Cell lines that were most sensitive to the drugs with respect to cell kill were also most sensitive to induction of sister chromatid exchanges for a given drug, and the assay based on sister chromatid exchange is therefore predictive of the relative sensitivity of these cells to the drugs used.

alpha-Difluoromethylornithine, an enzyme-activated, irreversible inhibitor of ornithine decarboxylase, inhibited the growth of both chloroethylnitrosourea-sensitive and -resistant 9L rat brain tumor cells in vitro. After 48 hr of treatment with 10 mM alpha-difluoromethylornithine, the putrescine and spermidine contents of both resistant and sensitive cells were less than 5% of control levels, but the spermine level was slightly elevated. The cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea, as measured by a colony-forming efficiency assay, was significantly increased in alpha-difluoromethylornithine-pretreated sensitive cells but not in resistant cells treated with this polyamine inhibitor. With the sister chromatid exchange assay, we found that alpha-difluoromethylornithine pretreatment increased 1,3-bis(2-chloroethyl)-1-nitrosourea-induced damage to chromosomes in sensitive but not in resistant cells.

The ability of various nitrosoureas to induce sister chromatid exchanges (SCEs) in 9L rat brain tumor cells was investigated. Treatment of cells for 1 hr with the alkylating and cross-linking agents 1,3-bis(2-chloroethyl)-1-nitrosourea or chlorozotocin produced concentration-dependent increases in SCEs; elevations above controls were detected at concentrations of 1 microM or more. Above 0.25 mM, the alkylating agent ethylnitrosourea produced a dose-dependent increase in SCEs. Treatment with the carbamoylating agent 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea did not induce SCEs. The maximum drug concentration at which SCEs are readily scored kills approximately 50% of cells. When accurate cell survival data in this dose range were obtained, a direct correlation between nitrosourea-induced cell kill, measured by a colony-forming efficiency assay, and SCE induction was found. Thus, analysis of the levels of SCE production may provide information about the efficacy of antineoplastic drugs.

The effects of hyperthermia (42 degrees C) on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-mediated DNA interstrand crosslink formation were investigated in 9L rat brain tumor cells using the technique of alkaline elution. When cells were treated with 60 microM BCNU for 1 hr at 37 degrees C and incubated for 6 hr in drug-free medium at 42 degrees C, there was a 50% increase in crosslinking; and when cells were treated at 42 degrees C and incubated at 37 degrees C, there was a 45% increase in crosslinking compared with the results for cells treated and incubated at 37 degrees C. When cells were treated and incubated at 42 degrees C, there was a 129% increase in DNA crosslinking. The same relative order of results was found for cell survival. These results suggest that hyperthermia can increase DNA interstrand crosslink formation and the consequent cell death through two independent mechanisms: an increase in the amount of initial alkylation because of the increased rate of hydrolysis of BCNU at higher temperatures, and the effect of heat on DNA structure that leads to an increase in the number of crosslinks formed.

To investigate the potential use of the sister chromatid exchange assay to analyze the heterogeneity of drug response among tumor cell subpopulations, mixtures of 1,3-bis (2-chloroethyl)-1-nitrosourea-sensitive (9L) and -resistant (9L2) rat brain tumor cells were treated in vitro with 2 microM 1,3-bis (2-chloroethyl)-1-nitrosourea. When data were plotted as histograms representing the number of cells vs sister chromatid exchanges/metaphase, two regions corresponding to the 9L and 9L2 populations were obtained. The approximate percentages of 9L and 9L2 in each mixture could be predicted from these histograms. While these results were obtained with a limited model chosen for its simplicity, they suggest that the sister chromatid exchange assay may be useful in the analysis of heterogeneity in drug sensitivity among cell subpopulations in a tumor.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU)-sensitive and BCNU-resistant 9L rat brain tumor cells were treated with BCNU at graded temperatures between 37 and 44 degrees C. The cytotoxic effects of hyperthermia alone on both cell lines were the same. Treating both cell lines with BCNU at temperatures above 37 degrees C caused a progressive increase in cell kill. All survival curves for drug-sensitive cells had shoulders followed by a region of exponential cell kill; dose enhancement ratios calculated at the 10% survival level ranged from 1.7 to 3.0. Survival curves for drug-resistant cells were exponential without a shoulder; dose enhancement ratios ranged from 3.3 to 8.4. For each cell line, a similar amount of the increased cell kill could be explained on the basis of the increases concentration of reactive species produced by hydrolysis of BCNU at elevated temperatures. The amount of cell kill that cannot be explained on this basis, however, suggests that factors other than an increase in the concentration of reactive species at higher temperatures are involved in the enhanced cell killing. Possible mechanisms include a heat-induced change in the structure of DNA chromatin and the effect of isocyanate deactivation of repair enzymes, both of which could lead to an increase in the number of crosslinks formed and therefore to an increase in cytotoxicity.

Cytotoxic effects of treating RIF-1, EMT6, and 9L tumor cell lines with cis-diamminedichloroplatinum(II) were measured with the sister chromatid exchange assay and compared to results obtained with the colony-forming efficiency assay. The greatest number of sister chromatid exchanges was induced in RIF-1 cells, fewer in EMT6 cells, and the least in 9L cells. Cell survival data obtained with the colony-forming efficiency assay paralleled data obtained with the sister chromatid exchange assay. These studies suggest that the sister chromatid exchange assay may be a useful method with which to determine the in vitro sensitivity of tumor cells to some antineoplastic agents.