Hugh F. English

Depletion of O6-alkylguanine-DNA alkyltransferase activity in mammalian tissues and human tumor xenografts in nude mice by treatment with O6-methylguanine.

SummaryWe have previously shown that exposure of cells in culture to O6-methylguanine significantly reduces their level of the repair protein, O6-alkylguanine-DNA-alkyltransferase (AGT), thus rendering cells more sensitive to the cytotoxic effects of chemotherapeutic chloroethylating agents. Experiments were carried out in mice to determine whether the AGT content of tissues and tumors could be reduced by in vivo treatment with O6-methylguanine. There was a dose-dependent decrease in AGT activity in liver tissues of CD-1 mice to 24% of basal levels after four hourly intraperitoneal injections of O6-methylguanine (110 mg/kg). Although the decline in AGT activity in the liver was reversible, the activity remained at 75% of basal levels for up to 25 h after the final injection. The effect of O6-methylguanine treatment on AGT activity was measured in mouse tissues as well as human colonic carcinoma tumors (HT29 and BE) grown in Swiss athymic nude mice. The activity in the liver, kidney, and spleen of these mice decreased to 33%–35% of control levels, whereas the activity in HT29 tumors was likewise diminished to 25% of control levels after four hourly injections of O6-methylguanine (100 mg/kg). There was no enhancement of the tumoricidal effectiveness of chloroethylating agents on the HT29 tumor after O6-methylguanine treatment, probably due to a disproportionately higher level of AGT in human tissue than in murine tissue. However, these studies suggest that O6-methylguanine can be given in vivo to examine the role of the AGT protein in protecting against the toxic and carcinogenic effects of alkylating agents.

Effect ofO6-benzylguanine on the response to 1,3-bis(2-chloroethyl)-1-nitrosourea in the Dunning R3327G model of prostatic cancer

The DNA-repair proteinO6-alkylguanine-DNA alkyltransferase is known to protect tumor cells from the antitumor effects of carmustine (BCNU). This repair protein was inactivated in Copenhagen rat prostate tumors by treatment withO6-benzylguanine in attempts to increase the effectiveness of BCNU therapy. The alkyltransferase activity in the liver, kidney, lung, and prostate of Copenhagen rats was 66, 37, 65, and 122 fmol/mg protein, respectively. The activity in the Dunning R3327G rat prostate tumor was found to be 129 and 126 fmol/mg protein from intact and castrated animals, respectively. The level of this protein remained low in the tissues and tumors of rats for up to 24 h and slowly began to rise at 36 h following an i. p. injection of 80 mg/kgO6-benzylguanine. Animal survival and body weight as well as tumor volumes were monitored in rats bearing prostate tumors in the flank area that had received no treatment,O6-benzylguanine alone, BCNU alone (5.5–60 mg/kg), or 80 mg/kgO6-benzylguanine 1 h prior to BCNU (5.5 mg/kg). WhenO6-benzylguanine was combined with BCNU therapy, there was a regression in tumor growth that was not observed in animals treated with an equal dose of BCNU alone. A similar regression in tumor growth was observed in animals treated with a higher dose of BCNU alone (45 mg/kg); however, this regimen was more toxic thanO6-benzylguanine plus BCNU (5.5 mg/kg) as determined by animal weight loss. The mean weight loss observed in animal treated with BCNU alone and in those given the combination was 24% and 6%, respectively. Histopathology revealed that animals receiving either BCNU alone or the combination had a decrease in all types of bone marrow cells, a loss of intestinal crypts, and a decreased number of lymphocytes in the spleen. The enhancement of the antitumor effect on BCNU by pretreatment withO6-benzylguanine supports a role for this therapy in the treatment of prostate cancer.

Kinetic and morphometric responses of heterogeneous populations of NMU-induced rat mammary tumor cells to hormone and antipolyamine therapyin vivo

The present experiments were designed to evaluatein vivo the differential sensitivity of tumor cell subpopulations to hormone and polyamine manipulations using the hormone-responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumor. NMU tumor bearing rats were randomly assigned to control, ovariectomy, α-difluoromethyl-ornithine (DFMO) administration (an inhibitor of polyamine biosynthesis), or combination treatment, and were sacrificed on day 2, 4, or 7. The proportion of different cells was estimated by morphometric analysis and their replicative activities by [3H]-thymidine autoradiography.In tumors of intact rats, the fractions of glandular, myoepithelial, and non-epithelial cells were 85.3 ± 2.2%, 4.7 ± 0.7%, and 9.9 ± 1.9%, respectively. Ovariectomy induced a similar time-dependent decline in the labelling indices of each cell type (from 5% to 1%). It also decreased the fraction of glandular cells (74.9 ± 4.5%), while increasing the fraction of myoepithelial (8.6 ± 1.9%) and non-epithelial (16.3 ± 3.2%) cells. DFMO exerted similar but more modest effects. DFMO-induced tumor regression was also inferior to that observed with ovariectomy. Combined ovariectomy and DFMO induced a faster and greater suppression of all labelling indices than the individual treatments, even though tumor regression was not superior to that produced by ovariectomy alone. Combination treatment also produced more profound morphologic changes, reducing the fraction of glandular cells to 64.4 ± 3.9% and increasing that of non-epithelial cells to 26.6 ± 4.4%. Ovariectomy and DFMO reduced height but not width of glandular cells, resulting in a modest decrease in cell volume. The combination treatment, however, significantly suppressed all three parameters.Cellular levels of polyamines were only modestly affected by the treatment used, thus raising doubts on their role as mediators, at least of ovariectomy-induced effects. Nevertheless, these results emphasize the sensitivity of different cell components of NMU tumors to combined hormone and anti-polyamine therapy with regard to kinetic and morphometric changes.

Androgen-primed chemotherapy experimental confirmation of efficacy

A current hypothesis suggests that androgen administration prior to chemotherapy (androgen priming) may potentiate tumor cytotoxicity in prostate cancer. The Dunning R3327G rat prostatic tumor model was used to test this concept experimentally. Control groups without priming included (1) intact untreated, (2) castrate alone and (3) castrate+ chemotherapy (cyclophosphamide, 30 mg/kg/day for 2 days with repeat cycle in 25 days- CTX). Two experimental groups received androgens, one before and one after chemotherapy. Treatment effect was monitored by quantitating tumor volume and animal survival. Control groups receiving castration and chemotherapy had a retardation of tumor growth and a prolongation of survival when compared to untreated animals. Androgen priming before but not after chemotherapy enhanced the degree of tumor suppression. With the androgen-priming protocol, all androgen-primed tumors had regressed, 3/6 tumors had disappeared and 3 were only palpable. At the same time point, tumors in all the other groups were actively growing and had volumes greater than the initial values (P less than 0.01). Median survival was significantly prolonged in primed animals 191 vs 40 days for untreated animals and 150 days for the nonprimed castration + chemotherapy animals (P less than 0.02). These findings have been repeated with several replicate experiments. These observations confirm the hypothesis that androgen priming can potentiate chemotherapy in an experimental system.