Synergistic interaction of ultrasonic shock waves and hyperthermia in the Dunning prostate tumor R3327-AT1.

Pulsed high-energy ultrasound shock waves (PHEUS), similar to those used for clinical lithotripsy, can deposit energy deep in tissue and thereby destroy the microvasculature of solid tumors. We investigated the potential of PHEUS, generated by an electromagnetic shockwave source (19 kV capacitor voltage, 1 Hz pulse frequency), as a local cancer-therapy modality alone and in combination with local tumor hyperthermia (43.5 +/- 0.1 degrees C, 30 min). Copenhagen rats transplanted with the anaplastic Dunning-prostate-tumor sub-line R3327-AT1 received 1000 PHEUS pulses, which delayed tumor growth by one tumor-doubling time (5 days). Histopathology revealed hemorrhage, disruption of tumor vasculature, and necrosis in the focus of the sound field. Bromodeoxyuridine (BUdR) incorporation was significantly lower in PHEUS-treated tumors than in controls. Dynamic magnetic resonance imaging (MRI) studies using gadolinium-DTPA as contrast agent showed a strong reduction of tumor perfusion after PHEUS treatment, although this effect was partly reversible within 3 days after PHEUS. While hyperthermia alone produced no significant delay in tumor growth, the combination of PHEUS and hyperthermia produced tumor-growth delay by 2 tumor-volume-doubling times. The maximum growth delay was achieved when PHEUS and hyperthermia were separated by 24 hr at the time of maximum perfusion reduction indicated by MRI. Thus, the cytotoxic effect of PHEUS was enhanced by hyperthermia in the anaplastic prostate tumor R3327-AT1 grown on Copenhagen rats in a synergistic manner, due to blood-flow reduction. In conjunction with other agents, such as hyperthermia, PHEUS might become a local cancer-therapy modality in solid tumors accessible to ultrasound.     

Treatment of Dunning R3327-AT rat prostate tumors with photodynamic therapy in combination with misonidazole

Fischer X Copenhagen rats bearing Dunning R3327-AT tumors were treated with hematoporphyrin derivative and red light (630 nm from an argon-driven dye laser) alone or in combination with the hypoxic cell radiosensitizer, misonidazole (MISO). In vitro studies had suggested that hypoxia might significantly decrease the cytotoxicity of photodynamic therapy (PDT) and labeling with [14C]MISO had revealed a significant fraction of viable hypoxic cells in this tumor. PDT alone resulted in a growth delay of 8.8 days but no tumor cures were observed. The administration of MISO (i.p. at 0.5 mg/g) 33 min prior to PDT resulted in an average growth delay of 15.2 days and tumor cures (local control at 33 days) in 20% of animals treated. If MISO at a similar dosage was administered 30 min after PDT an average growth delay of 16.3 days was measured and tumor cure was observed in 70% of the animals treated. These results suggest that the hypoxic cell fraction in R3327-AT tumors might be a limitation to their curability by PDT. The addition of MISO and/or other hypoxic cell cytotoxic agents to PDT procedures may provide an effective means of treating PDT-resistant hypoxic cells in solid tumors.     

Tumor oximetry: demonstration of an enhanced dynamic mapping procedure using fluorine-19 echo planar magnetic resonance imaging in the Dunning prostate R3327-AT1 rat tumor

PURPOSE: We have developed an enhanced approach to measuring regional oxygen tension (pO(2)) dynamics in tumors. The technique is demonstrated in a group of 8 Dunning prostate rat tumors (R3327-AT1) with respect to respiratory challenge. METHODS AND MATERIALS: Hexafluorobenzene was injected directly into the tumors of anesthetized rats. (19)F nuclear magnetic resonance echo planar imaging relaxometry was performed to obtain maps of regional tumor oxygenation under baseline conditions and when the inhaled gas was changed to oxygen or carbogen. RESULTS: Sequential pO(2) maps required 8 min, with a typical precision of 1-3 torr at 30-100 individual regions across a tumor. When rats breathed 33% oxygen, distinct heterogeneity was observed for baseline oxygenation in each tumor with pO(2) values ranging from hypoxic to greater than 100 torr. Larger tumors showed significantly lower baseline pO(2). Respiratory challenge with oxygen or carbogen produced significant increases in tumor oxygenation with a close correlation between the response to each gas at individual locations. Regions of both small and large tumors responded to respiratory challenge, but the rate was generally much faster in initially well-oxygenated regions. CONCLUSIONS: Regional pO(2) was assessed quantitatively and the response of multiple individual tumor regions observed simultaneously with respect to interventions.     

Repression of thermotolerance in Dunning R3327 prostate carcinoma cells by 2-deoxy-glucose

The transient addition of the cytosolic energy depletor 2-deoxy-glucose to cultures of rat prostate carcinoma cells blunted the induction of Hsp70 protein following exposure to elevated temperatures in a manner that appeared to parallel its effects on energy metabolism. While the reduction in stress-induced heat-shock protein expression by treatment with 2-deoxy-glucose had no effects on the acute loss of cellular viability after exposure to heat, the acquisition of thermotolerance in response to a conditioning stimulus was specifically repressed. Therefore, 2-deoxy-glucose will be a useful tool in the investigation of mechanisms that mediate immediate versus chronic responses to cellular stress, including the specific roles played by members of the heat-shock protein family of proteins. These results might have important implications in the design of protocols for the hyperthermic treatment of tumours.     

The response of Dunning R3327 prostatic adenocarcinoma to IL-2, histamine and radiation.

A syngeneic, androgen-sensitive Dunning R3327 rat prostatic adenocarcinoma was transplanted bilaterally in the flanks of male Copenhagen Fisher rats. Approximately 3 months after implantation, when the tumours had a median volume of 150 mm3, one group of rats was treated with histamine alone (4 mg kg(-1) subcutaneously on week days), another group with human recombinant interleukin 2 (IL-2) alone (425 IU kg(-1) continuous infusion) and a third group with both histamine and IL-2 during 6 weeks. Tumours on one flank were irradiated (6 Gy once daily for 3 days to a total dose of 18 Gy) beginning 1 week after the onset of treatment with histamine and/or IL-2. The contralateral tumour served as the intra-animal control. The tumour volumes were determined weekly. The growth curves showed that all three drug treatments were effective in delaying growth, but when used individually did not cause tumour shrinkage. Radiation was the most effective single agent, but when used alone the shrinkage did not occur until 2 weeks after irradiation. When combined with the drugs, more rapid and extensive growth delay and/or shrinkage was seen. The growth curves showed clear differences between the different treatments. The combination of the three agents was the most effective of all. The most striking difference between radiation alone and radiation plus biotherapy was the time at which a tumour response was detectable. Thus, active biotherapy alone and especially in a combination with histamine and radiotherapy warrants further investigation as a potential therapeutic approach to prostate cancer.     

Repression of thermotolerance in Dunning R3327 prostate carcinoma cells by 2-deoxy-glucose.

The transient addition of the cytosolic energy depletor 2-deoxy-glucose to cultures of rat prostate carcinoma cells blunted the induction of Hsp70 protein following exposure to elevated temperatures in a manner that appeared to parallel its effects on energy metabolism. While the reduction in stress-induced heat-shock protein expression by treatment with 2-deoxy-glucose had no effects on the acute loss of cellular viability after exposure to heat, the acquisition of thermotolerance in response to a conditioning stimulus was specifically repressed. Therefore, 2-deoxy-glucose will be a useful tool in the investigation of mechanisms that mediate immediate versus chronic responses to cellular stress, including the specific roles played by members of the heat-shock protein family of proteins. These results might have important implications in the design of protocols for the hyperthermic treatment of tumours.     

Radiation sensitivity of Copenhagen rat prostatic carcinoma (R3327-AT and R3327-MATLyLu)

The radiosensitivity of two variants of the Dunning Copenhagen rat prostatic tumor R3327 was investigated. The R3327-AT variant, which is a poorly differentiated anaplastic, fast-growing tumor, was irradiated both in vivo and in vitro. Following irradiation, monodispersed cells were plated in vitro and colonies were counted after 7 days. The survival curve of R3327-AT cells irradiated in vivo showed an initial shoulder (Dq-value 0.97 Gy), followed by two exponential parts. The D0-value for the first part of the curve (0-10 Gy) was 2.76 Gy and for the second part of the curve (greater than 10 gy) 9.05 Gy. Extrapolation of the second part of the curve to the Y-axis indicated that the proportion of more radioresistant cells was about 10%. The survival curve for R3327-AT cells irradiated in vitro also suggested the presence of a radioresistant subpopulation, although the proportion was lower (about 3%). This difference might be due to the presence of an hypoxic fraction in the tumors irradiated in vivo, but not in vitro. Tumor cells from the R3327 tumor variant metastatic to lymph nodes and lungs (R3327-MATLyLu), were irradiated in vitro. The radiation effect was evaluated by in vitro colony formation in agar and by in vivo lung colony assay. The colony formation in agar yielded a D0-value of 1.09 Gy. No radioresistant subpopulation was identified in this variant. A similar radiosensitivity was observed by the in vivo lung colony assay (D0 1.39 Gy). The mean inactivation dose calculated for R3327-AT cells (3.45 Gy) was significantly higher than for the metastatic variant (2.00 Gy).     

Supraadditive apoptotic response of R3327-G rat prostate tumors to androgen ablation and radiation

: Androgen ablation is often combined with radiation in the treatment of patients with prostate cancer, yet, the optimal sequencing and the mechanisms governing the interaction are not understood. The objectives were to determine if cell killing via apoptosis is enhanced when the combined treatment is administered and to defined the relationship of changes in this form of cell killing to tumor volume growth delay.: Dunning R3327-G rat prostate tumors, grown inthe flanks of Copenhagen rats, were used at a volume of approximately 1 cc. Androgen ablation was initiated by castration, and androgen restoration was achived with 0.5 cm silastic tube implants containing testosterone. ⁶⁰Co was used for irradiation. The terminal deoxynucleotidyl transferase (TUNEL) histochemical assay was used to quantify apoptosis.: Tumors from intact and castrate unirradiated control rats had average apoptotic indices (percent of apoptotic cells) of 0.4 and 1.0%, respectively. The apoptotic index varied only slightly over time (3 h to 28 days) after castration (range 0.75-1.43%). Irradiation of intact rats to 7 Gy resulted in a peak apoptotic response at 6 h of 2.3%. A supradditive apoptotic response was seen when castration was initiated 3 days prior to 7 Gy radiation, with peak levels of about 10.1%. When the radiation was administered at increasing times beyond 3 dats after castration, the apoptotic response gradually diminished and was back to levels seenin intact rats by 28 days after castration. Tumor volume growth delay studies were consistent with, but not conclusive proof of, a supraadditive effect when the combination was used.: A supraaddtive apoptotic response was seen when androgen ablation and radiation were used to treat androgen sensitive R3327-G rat prostate tumors. This supraadditive effect was dependent on the timing of the two treatments. Further studies are required to more fully define the optimal timing and administration of androgen ablation and radiation.     

Systematic investigation into interaction of ionizing radiation and doxorubicin in the Dunning R3327G prostatic adenocarcinoma model

The interaction of doxorubicin and radiation has been systematically studied in the Dunning R3327G prostatic adenocarcinoma, the preeminent animal model for human prostatic cancer. Subcutaneous tumors (produced by injection of 10(7) cells) were treated when about 1 cm3 in volume (19-22 days postimplant). Each modality was used at 1 of 3 dose levels; 2, 4, and 9 mg/kg for doxorubicin; and 5, 15, and 25 Gy for radiation. Single treatment with each agent was combined, in both sequences and five delay times (0.5, 12, 24, 48, and 120 hr) between agents. Growth of individual tumors was fit to a quadratic exponential growth model which was solved for the growth delay and growth rate at twice initial volume. Analysis of variance identified significant interactions for doxorubicin and radiation (due to drug toxicity), sequence and delay, and sequence and radiation, in addition to the four factors individually. The effect on the tumor of combined doxorubicin and radiation is basically additive. Sequence and delay are important in overall tumor control.     

Differential oxygen dynamics in two diverse Dunning prostate R3327 rat tumor sublines (MAT-Lu and HI) with respect to growth and respiratory challenge

PURPOSE: Since hypoxia may influence tumor response to therapy and prognosis, we have compared oxygenation of tumors known to exhibit differential growth rate and tissue differentiation. METHODS AND MATERIALS: Regional tumor oxygen tension was measured using 19F nuclear magnetic resonance echo planar imaging relaxometry of hexafluorobenzene, which provided dynamic maps with respect to respiratory intervention. Investigations used two Dunning prostate R3327 rat tumor sublines: the fast growing, highly metastatic MAT-Lu and the moderately well-differentiated, slower growing HI. RESULTS: Both sublines showed significantly higher oxygen tension in smaller tumors (<2 cm(3)) than in larger tumors (>3.5 cm(3)). Pooled data showed that MAT-Lu tumors exhibited greater hypoxia compared with the size-matched HI tumors (p < 0.0001). Respiratory challenge (oxygen or carbogen) produced significant increases in mean pO(2) for tumors of both sublines (p < 0.0001). However, initially hypoxic regions displayed very different behavior in each subline: those in the HI tumors responded rapidly with significant elevation in pO(2), while those in the MAT-Lu tumors showed little response to respiratory intervention. CONCLUSIONS: These results concur with hypotheses that hypoxia is related to tumor growth rate and degree of differentiation. Under baseline conditions, the differences were subtle. However, response to respiratory intervention revealed highly significant differences, which, if held valid in the clinic, could have prognostic value.     

Hormonally responsive versus unresponsive progression of prostatic cancer to antiandrogen therapy as studied with the Dunning R-3327-AT and -G rat adenocarcinomas

The present study has compared the response to antiandrogen therapy of the serially transplantable Dunning R-3327-AT (hereafter called AT) versus Dunning R-3327-G (hereafter called G) rat prostatic adenocarcinoma. Castration or chemical antiandrogen therapy (i.e., cyproterone acetate and diethylstilbestrol) of rats bearing established AT or G tumors results in neither regression of tumor volume nor a cessation of the continuous growth of either tumor. By these criteria, both the AT and G tumors progress following antiandrogen therapy. For the AT tumor, this progression is completely unresponsive to hormonal therapy, and thus such therapy does not increase survival of AT tumor-bearing rats. The AT tumor is therefore an example of hormonally unresponsive progression. In direct contrast, while the G tumor likewise progresses following antiandrogen therapy, this therapy does induce a 1.8-fold decrease in the subsequent growth rate of the G tumor. This positive response during progression of the G tumor results in a 78% increase in the survival of G tumor-bearing rats treated with antiandrogen therapy. The G tumor is therefore an example of hormonally responsive progression. These results indicate neither that prostatic cancers which do not regress or cease growing following antiandrogen therapy can necessarily be considered hormonally unresponsive nor that antiandrogen therapy of such tumors has been completely ineffective, since, as shown in the present study, such progression can be of either a hormonally unresponsive or a responsive type. Regardless of which type of progression occurs, however, additional therapy is required to further increase survival. The present study demonstrates that such additional therapy should probably not include the subsequent use of pharmacological doses of exogenous androgen, since, depending on the type of progression, such treatments can actually decrease survival.     

Beneficial effects of androgen-primed chemotherapy in the Dunning R3327 G model of prostatic cancer

The objective of this study was to test the hypothesis that androgen administration prior to chemotherapy (androgen priming) may potentiate tumor cytotoxicity in hormone-responsive prostate cancer. Accordingly, six groups of Copenhagen rats bearing small (i.e., 40-mm3 median volume) Dunning R3327 G tumors were left untreated or received castration, chemotherapy, or a combination of the two, with or without androgen priming. Groups without priming included: intact untreated, castrate alone, intact plus chemotherapy, and castrate plus chemotherapy (cyclophosphamide, 30 mg/kg/day, for 2 days, with repeat cycle in 24 days) (Cx). To specifically evaluate the effect of androgen priming on Cx cytotoxicity, two additional castrate groups were studied. One received testosterone propionate (4 mg/kg/day) for 2 days prior to Cx and the other after Cx. Treatment effect was evaluated by quantitating tumor volume as well as animal survival to an ethically allowable, maximal tumor burden. As expected, castration and Cx produced a retardation of tumor growth and prolongation of survival when compared to untreated animals. The addition of androgen priming prior to but not after Cx enhanced the degree of tumor suppression. Specifically, 26 days after the second Cx cycle, all androgen-primed tumors had regressed; 70% of tumors had disappeared and those remaining were barely palpable. At this same time point, tumors in all the other groups were actively growing and had volumes greater than initial values (P less than 0.01). Although tumor regrowth occurred, median survival for the androgen-primed group was significantly prolonged, to 186 days versus 39 days (P less than 0.01) for untreated animals and 153 days for the non-primed castrate plus Cx animals (P less than 0.01). These data suggested that androgen priming potentiates the effects of Cx in castrate animals bearing R3327 G tumors.     

In vivo effects of high-intensity ultrasound on prostatic adenocarcinoma Dunning R3327.

High-intensity ultrasound has been used to treat Dunning R3327 prostatic adenocarcinoma implanted s.c. in Fischer Copenhagen rats. Focused ultrasound was generated with a 1-MHz transducer and energy was provided by a 7.5-kW power amplifier. Seventy-four rats were treated using two different sublines of Dunning tumor. Study 1 dealt with 49 rats with the Mat-Ly-Lu subline, treated with acoustic intensities ranging from 300 to 2750 W/cm2. Of the 49 rats in Study 1, 30 had complete tumor necrosis and 19 had no effect; of the 30 who had complete local tumor necrosis, 14 had local relapse, 9 had distance metastases to lung and nodes without local occurrence, and 7 remained free of tumor and were still alive 12 months after treatment. In Study 2, 25 rats with AT2 subline were treated with an intensity of 820 W/cm2. Similarly for Study 2, there was complete local tumor necrosis in 24 of 25 animals, with local regrowth in 7 of 24 and no recurrence of metastasis in the remaining 16 after a follow-up of 3 months. These results suggested that high-intensity focused ultrasound could be useful for the treatment of small localized cancerous tumors such as low-grade prostate carcinoma.     

Proliferative response of the Dunning R3327H experimental model of prostatic adenocarcinoma to conditions of androgen depletion and repletion

Administration of androgen to the castrate rat elicits a pronounced wave of proliferative activity in the ventral prostate gland. We wished to determine if this phenomenon could also be demonstrated in malignant prostate tissue. An experimental protocol consisting of 12 days of androgen depletion induced by castration followed by 7 days of androgen repletion was utilized in animals bearing the androgen-dependent Dunning R3327H prostatic adenocarcinoma. Repletion of androgen levels was achieved by daily s.c. injection of testosterone or dihydrotestosterone at 8, 16, or 32 mg/kg of animal body weight. The proliferative response of the tumor to conditions of depletion as well as repletion on Days 3, 5, and 7 was determined using [3H] thymidine autoradiography, quantitative morphometry, and flow cytometric analysis. The autoradiographic and flow cytometric data were complementary and indicated that androgen depletion caused a slight reduction in the percentage of S-phase nuclei. Repletion initiated a highly reproducible, significant increase in both the S-phase compartment as well as tritiated thymidine incorporation into DNA, and of the days quantitated, the greatest values were obtained on Day 3. The response patterns were nearly identical for both testosterone and dihydrotestosterone, and no significant differences were detected among the doses used. Quantitation of the autoradiographs revealed a striking disparity in the response of the cell types. The labeling indices of nonepithelial cells increased only minimally during repletion, whereas the epithelial cells responded consistently and reached levels 2- to 4-fold over intact values. These data indicate that protocols of androgen depletion/repletion have the capacity to elicit a significant wave of cell proliferation. These manipulations support the feasibility of transiently increasing the number of cancer cells in S phase as a means of potentiating cytotoxic chemotherapy for treatment of adenocarcinoma of the prostate.     

R3327 prostate adenocarcinoma clonogenic cells: epithelial properties and hormone response

Cell colonies derived from the clonogenic tumor cell [colony-forming cell, prostate adenocarcinoma (CFC-PA)] assayed in vitro from the R3327 rat prostate adenocarcinoma demonstrate prostate acid phosphatase activity when assayed histochemically and convert testosterone to stanolone. The number of CFC-PA/10(4) cells plated in steroid-free cultures was increased following the addition of testosterone or stanolone and decreased following the addition of 17 beta-estradiol. The decreased rate of growth of the R3327 tumor in castrated male inbred Copenhagen rats when compared to the growth measured in normal (intact) male and female inbred Copenhagen rats was reflected in a large decrease in the number of CFC-PA/10(4) cells plated from tumors grown in castrated male rats when compared to the values obtained from tumors that were grown in normal male and female rats. Furthermore, the replacement of fetal calf serum with normal male or castrated male rat serum resulted in little change in CFC-PA/10(4) cells plated in cultures established from tumors grown in castrated rats, although significant increases in CFC-PA were observed in cultures established from tumors grown in normal male or female rats.     

Tumor progression in serial passages of the Dunning R3327-G rat prostatic adenocarcinoma: growth rate response to endocrine manipulation

Serial passages of the poorly differentiated, androgen-sensitive R3327-G prostatic adenocarcinoma were used to study the progressive changes that occur in tumor growth rate and androgen sensitivity. Different in vivo transplant generations (21st to 28th) were compared. The tumor doubling and animal survival times resulting from the implantation of the 21st to 22nd generation (21-22G) tumor cells in intact male rats were significantly greater than those resulting from the implantation of 23-28G tumor cells. The most dramatic difference between early (21-23G) and late (26-28G) tumor generations, however, was in androgen sensitivity. The 26-28G tumors displayed androgen sensitivity only when implanted into animals castrated 2 to 7 days previously. Tumors grown in the pretreated castrates grew at a significantly slower rate than those in intact rats and the pretreated castrates had longer survival times than the intact rats. When 26-28G tumors were allowed to grow in intact rats to approximately 1 cu cm and then the rats were castrated, no significant difference in the growth rate between these tumors and tumors grown in intact rats was observed. In contrast, the androgen sensitivity of 21-23G tumors could be demonstrated, regardless of whether treatment was started before or after implantation. The fact that androgen sensitivity was still evident under certain conditions in late-generation R3327-G tumors demonstrates that the basic mechanism involving androgen response was still present, although functioning at a much reduced level.     

The early supra-additive apoptotic response of R3327-G prostate tumors to androgen ablation and radiation is not sustained with multiple fractions

PURPOSE: The treatment of R3327-G tumor-bearing rats with androgen ablation (AA) via castration results in a supra-additive increase in apoptosis when 2-8 Gy gamma-irradiation (RT) is given as a single dose 3-14 days afterwards. We report here the dose response and effect of multiple fractions on this supra-additive apoptotic response. MATERIALS AND METHODS: Dunning R3327-G tumors were grown in the flanks of Copenhagen rats and the experiments were initiated at a tumor volume of 1.0-1.5 cc. Androgen ablation was achieved by castration 3 days prior to gamma-irradiation. Apoptosis was measured with a terminal deoxynucleotidyl transferase dUTP-biotin nick end-labeling assay 6-h after RT, unless otherwise specified. RESULTS: The dose response of the supra-additive apoptotic response was assessed by irradiating castrated animals with single doses of 2, 4, 8, or 16 Gy (n = 5 per group); tumor cell apoptosis at 6-h following irradiation was 2.4%+/-0.7% (+/- SEM), 4.2%+/-0.8%, 6.5%+/-1.4%, and 1.6%+/-0.3%, respectively. The RT only and AA only controls had < 1% apoptosis. The effect of fractionated RT on apoptosis was investigated to determine if the supra-additive apoptotic response was sustained with repeated 2-8 Gy fractions. When tumor-bearing animals were treated with repeated daily 2-Gy fractions, there was a reduction in the level of the supra-additive apoptotic response. After five 2-Gy fractions at 24-h intervals, apoptosis in the combined treated tumors was at levels seen in the AA controls. This raised the possibility that more than 24 h are required for recovery of the high supra-additive apoptotic levels seen after one fraction. When the interfraction interval was extended to 96 h, there was no significant increase in apoptosis over the additive effect of AA and RT. Although there was a decline in supra-additive apoptosis with repeated fractions, a dose response for tumor growth delay was evident for RT alone using 2.5-Gy fractions. Moreover, the combination of AA + fractionated RT resulted in a supra-additive enhancement in tumor growth delay to 5 cc. CONCLUSION: The early supra-additive apoptotic response from AA and single fraction radiation is not seen at high single fraction doses and is not sustained with repeated fractions. Therefore, the classical apoptotic response that occurs within 24 h of irradiation is not likely to be the main mechanism responsible for any clinical benefit seen with this combination.     

Growth-related elevations of DNA topoisomerase II levels found in Dunning R3327 rat prostatic adenocarcinomas

The relationship between DNA topoisomerase II expression and mammalian cell proliferation has been evaluated by determining enzyme levels in normal and neoplastic rat prostate tissues. By activity assay and by immunoblot analysis using anti-topoisomerase II antiserum, topoisomerase II levels were found to be elevated in both the Dunning R3327-H and the Dunning R3327-G rat prostatic adenocarcinomas over levels assayed in the normal rat dorsal prostate. Immunohistochemical studies using the antitopoisomerase II antiserum revealed that a greater fraction of nuclei contained detectable levels of topoisomerase II in tissue sections prepared from each of the Dunning tumors than in rat dorsal prostate tissue sections. The Dunning R3327-H and R3327-G tumors grow at different rates in vivo (J. T. Isaacs and D. S. Coffey, Clin. Oncol., 2: 479-498, 1983). When measured topoisomerase II levels were compared to known growth parameters for each of the tissues studied, topoisomerase II expression was found to be correlated with tissue growth rate.     

Effect of the preestrogen 4-androstene-3,17-dion-19-al on the Dunning R3327 prostatic adenocarcinoma

The purpose of this pilot study was to determine if biogenetic precursors of estrone such as 4-androstene-3,17-dion-19-al, which is virtually devoid of thrombotic potential as well as androgenic and uterotrophic activity, could replace estrogen in the treatment of the hormone-sensitive Dunning R3327 prostatic adenocarcinoma in the male Copenhagen rat. If such were the case, the way would be open to an improved form of palliative therapy of prostatic cancer with the potential for decreased estrogenic side effects and cardiovascular complications. To this end, the R3327 tumor was transplanted (Day 0) into the flank of 10-week-old male Copenhagen rats, and treatment was begun 20 weeks later at which time the tumors reached a mean volume of 2160 cu cm. In addition to 4-androstene-3,17-dion-19-al (1 and 10 mg/day), diethylstilbestrol (33 micrograms/day) and 17 beta-estradiol (3.3 and 33 micrograms/day) were studied (daily for 60 days). At 1 mg/day, 4-androstene-3,17-dion-19-al produced a 43% inhibition of tumor growth (Day 203) while, in the 10 mg/day group, a 72% inhibition of tumor growth was measured on Day 196 (roughly equivalent to that produced by estradiol at 3.3 micrograms/day), with a 50% inhibition on Day 231. It is concluded that the tumor-inhibiting activity of 4-androstene-3,17-dion-19-al, coupled with its very low thrombotic potential, indicated that orally active analogues of this steroid may offer advantages over estrogens in the palliative treatment of prostatic cancer.     

Effect ofO 6-benzylguanine on the response to 1,3-bis(2-chloroethyl)-1-nitrosourea in the Dunning R3327G model of prostatic cancer

The DNA-repair proteinO ⁶-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 withO ⁶-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/kgO ⁶-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,O ⁶-benzylguanine alone, BCNU alone (5.5–60 mg/kg), or 80 mg/kgO ⁶-benzylguanine 1 h prior to BCNU (5.5 mg/kg). WhenO ⁶-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 thanO ⁶-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 withO ⁶-benzylguanine supports a role for this therapy in the treatment of prostate cancer.Abbreviations AGT O ⁶-alkylguanine-DNA alkyltransferase - BCNU 1,3-bis(2-chloroethyl)-l-nitrosourea