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Chromosome analysis was carried out on 22 patients with Crohn's disease, taking part in a double-blind clinical trial of metronidazole and sulphasalazine. The patients were randomized into 2 groups, one beginning on metronidazole 0.8 g daily, the other on sulphasalazine 3 g daily, and after 4 months' treatment changing to the other drug for another 4 months' treatment. Sampling for chromosome analysis was carried out at 0, 1, 4, 5 and 8 months of treatment. Neither metronidazole nor sulphasalazine produced any significant increase in the individual types of chromosome aberrations from month 0 to 4. There was a slight but not significant increase in the total number of aberrations after sulphasalazine treatment but not after metronidazole treatment. A significant increase in the number of chromatid and isochromatid breaks was found after the combined treatment, i.e. from month 0 to month 8, when the means for all 22 patients were analysed. However, metronidazole at a daily dose of 0.8 g for four months did not increase the level of chromosomal aberrations in Crohn's patients.

Estramustine is an antimicrotubule agent that is effective against prostate cancer when used in combination with other microtubule-binding drugs. It is a derivative of estradiol and has a nitrogen mustard group attached via an intervening carbamate group. The molecular dimensions published for estramustine from crystal structure analyses (Mol. Pharmacol. 41∶569, 1992) indicate that the carbamate group modifies the mustard group by giving considerable double-bond character to the C-N bond. As a result the mustard group cannot form an active aziridine ring and therefore does not show the expected alkylating function. The substitution at O(3) of the aromatic A ring of the steroid moiety has also modified its activity as a steroid. Geometric data are presented here on a compound in which the two chlorine atoms of the mustard group of estramustine are replaced by hydroxyl groups. The question was, why does the dihydroxy derivative not show biological activity when chlorine atoms do not appear to be activated in estramustine itself? A comparison of the molecular geometries of the two compounds shows that the dimensions of the carbamate group are similar in both compounds. Therefore it appears that it is the extensive hydrogen-bonding capability of the dihydroxy compound that destroys its estramustine-like activity. In crystals of both compounds there is a hydrogen bond between O(17) -H and O(19) of another molecule, but the dihydroxy compound can form two more hydrogen bonds. This may possibly prevent it from reaching the site of action of estramustine or, if it does reach that site, cause it to behave differently.

Malignant cells possess a high degree of proteolytic activity in which the plasminogen activator system plays an important role. An increased expression of urokinase type plasminogen activator (uPA) is of significance for degradation of the extracellular tumor matrix, facilitating invasiveness and growth. Inhibition of the active site of uPA makes it possible to evaluate the significance of uPA in tumor growth. We report here experiments on a uPA-producing human prostate xenograft (DU 145) using a competitive inhibitor of uPA, p-aminobenzamidine. In vitro experiments with DU 145 cells showed that p-aminobenzamidine caused a dose-dependent inhibition of uPA activity. DU 145 cells were inoculated s.c. in SCID mice and, once tumors were established, treatment with p-aminobenzamidine added to drinking water was started and lasted for 23 days. Mice receiving 250 mg/kg/day of p-aminobenzamidine showed a clear decrease in tumor-growth rate compared to the non-treated mice, resulting in 64% lower final tumor weight. In addition, uPA-antigen levels in the membrane fractions of DU 145 tumors from p-aminobenzamidine-treated mice were found to be decreased by 59%. We also show that p-aminobenzamidine has an anti-proliferative effect in cell culture at low cell number, correlating with a dose-dependent decrease in uPA production. In conclusion, we show that a low-molecular-weight uPA-inhibitor, p-aminobenzamidine, has a growth-inhibitory effect on a solid uPA-producing tumor.

Prostate cancer has a slow growing noninvasive phase, but, in general, is invasive on diagnosis. An initial step in the invasion of surrounding normal tissue is the activity of proteolytic enzymes such as components of the plasminogen activator system (PA). In cell culture, the primary human prostate cancer cell line 1013L expressed no urokinase type-PA (uPA), while DU 145, a cell line derived from a metastatic lesion, expressed high levels of uPA. The DU 145 cells grew easily as xenografts but the establishment of 1013L in the SCID mice was possible only with the aid of a gelatin sponge (Spongostan). The latency period was 42-64 days, followed by a slow growth phase before a fast growth phase occurred. This fast growth phase was characterized by rapid degeneration of tumor tissue, while high proliferation occurred around the blood vessels. On serial transplantation of tumor material, the growth pattern was similar. Furthermore, the 1013L tumor was encapsulated by connective tissue and no invasiveness could be detected. We found that 1013L tumor homogenates had hardly detectable levels of uPA, i.e., 300-fold lower than we found in the invasive prostate xenograft DU 145. In addition, no expression of uPA was found in the plasma of 1013L tumor-bearing mice whilst uPA antigen was detected in the plasma of DU 145 tumor-bearing mice. In conclusion, the 1013L cell line, which exhibits a nonaggressive pattern, could be a good model for studying progression of prostate cancer to a more aggressive phenotype in vivo and in vitro.

The present study was carried out with the 10 known or suspected spindle poisons of the Commission of the European Communities program to study aneuploidy induction. We have investigated these substance of the assembly of isolated bovine microtubules at 10, 100 and 1000 μM and studied morphology by electron microscopy. The substances could be grouped into two categories, strong and weak inhibitors. Colchicine, vinblastine and thiomerosal were strong inhibitors; cadmium chloride, thiabendazole, chloral hydrate, hydroquinone, diazepam and econazole were weak inhibitors, The latter three causing aberrant forms visible on electron microscopy. Pyrimethamine did not inhibit the assembly of microtubules, but produced aberrant forms.

A variety of cell biological, pharmacological, crystallographic and clinical approaches have indicated that the antimitotic drug estramustine has interesting and unusual properties. Although designed as an alkylating agent, the marked stability of the carbamate linkage to the steroid carrier molecule prevents the formation of alkylating intermediates. The affinity of the parent molecule for microtubule associated proteins and the concomitant antimicrotubule activity have cytotoxic consequences in tumor cells. Both preclinical and clinical studies of estramustine in combination with other antimicrotubule agents have shown that this approach has great potential to achieve therapeutic advantage, especially in disease states such as hormone refractory prostate cancer.

(1990). Mechanisms of Action and Clinical Uses of Estramustine. Cancer Investigation: Vol. 8, No. 3-4, pp. 375-380.

We describe a novel syngeneic "pseudo-orthotopic" in vivo model of prostate cancer progression. Our model uses the dorsal skinfold chamber technique with fluorescence video microscopy and TRAMP-C2 tumor cells. The cells were transfected with a histone H2B-GFP fusion protein, permitting real-time measurement of tumor size, as well as mitotic and apoptotic indices. To generate a "pseudo-orthotopic" milieu, pieces of prostate tissue (10-15 mm2) from donor mice were implanted into the chambers of C57BL/6 mice. The prostate tissue grafted into the chambers retained its native vasculature, as determined by transplantation of prostate tissue from GFP transgenic mice. TRAMP-C2 prostate cancer tumor spheroids (25,000 cells) were implanted in the chamber. Without prostate tissue, TRAMP-C2 prostate tumors were poorly angiogenic, displayed low mitotic and apoptotic indices (0.7 x 10(-4)), and no significant tumor growth could be detected. TRAMP-C2 tumors growing on transplanted prostate tissue in the chamber on the other hand had mitotic indices in the order of 1.6 x 10(-4) and apoptotic indices in the order of 0.8 x 10(-4). Furthermore, tumors with stroma were highly angiogenic, and were fully vascularized within 7-10 days. During a 4-week observation period, the number of tumor cells increased by nearly 300%. We used the model to study the effects of surgical castration. The most profound response was a rapid vascular regression of the tumor vasculature. Castration also increased apoptotic indices within the tumor without significant changes in mitosis. This model may be utilized for the rapid analysis of new therapeutic candidates against prostate cancer.

BACKGROUND The combination of paclitaxel, oral estramustine phosphate (EMP), and carboplatin (TEC) has shown antitumor activity in patients with castrate, metastatic prostate carcinoma. To improve the therapeutic efficacy and reduce the toxicity of TEC, the authors substituted intravenous (i.v.) EMP for oral EMP based on single-agent studies demonstrating an improved safety profile with i.v. EMP. METHODS Patients with progressive, castrate, metastatic prostate carcinoma were treated with up to 6 4-week cycles of i.v. EMP (500–1500 mg/m2 per week), paclitaxel (100 mg/m2 per week), and carboplatin (target area under the curve = 6 mg/mL every 4 weeks). RESULTS Thirty patients were treated in 6 dose cohorts. Deep venous thrombosis occurred in 5 of 30 patients (17%). Other common Grade 3/4 (according to National Cancer Institute Common Toxicity Criteria) toxicities included hepatic toxicity (23%) and leukopenia (24%). Posttherapy prostate specific antigen declines > 50% were seen in 18 of 30 patients (60%), and declines > 80% were seen in 15 of 30 patients (50%). Eleven of 17 patients (65%) with measurable soft tissue disease achieved a partial response. Four of 27 patients (15%) with osseous metastases demonstrated improvement on bone scan. CONCLUSIONS Intravenous EMP was administered safely with paclitaxel and carboplatin and produced clinical outcomes similar to the outcomes achieved with the TEC regimen. Substitution of i.v. EMP for the oral formulation was found to result in a lower incidence of severe nausea but increased hepatic toxicity. Cancer 2003. © 2003 American Cancer Society.

The purpose is to determine a safe weekly dose of i.v. estramustine phosphate (EMP) to combine with weekly paclitaxel and monthly carboplatin in patients with advanced prostate cancer.Patients with advanced prostate cancer (castrate and noncastrate) were administered escalating doses of weekly 1-h infusion of i.v. EMP (500-1000-1500 mg/m(2)) in combination with weekly paclitaxel (100 mg/m(2) over 1 h) and i.v. carboplatin (area under the curve 6 mg/ml-min every 4 weeks). Four weeks of therapy were considered one cycle. In the first three cohorts, EMP was given i.v. 3 h before paclitaxel. Cohorts 4 and 5 reversed the administration order: EMP (doses 1000-1500 mg/m(2)) was given immediately after the end of paclitaxel infusion. Plasma levels of EMP and its metabolites, estramustine and estromustine, were monitored at time 0, at 120 min, and approximately at 20, 21, and 168 h from the start of EMP infusion. Paclitaxel concentrations were determined at basal (0), 30, 60, 90, and 120 min and 18 h after the start of paclitaxel infusion, and a concentration-time curve was estimated. Pharmacokinetic evaluation was performed in cycles 1 and 2 during the first week of therapy.Nineteen patients were entered on the initial three dose levels (cohorts 1-3). Dose-limiting transient hepatic toxicity was encountered in cohort 3 (EMP = 1500 mg/m(2)). An additional 13 patients were treated with paclitaxel (100 mg/m(2)) first, followed by i.v. EMP at 1000 mg/m(2) (cohort 4), and 1500 mg/m(2) (cohort 5). No dose-limiting toxicities were seen, and cohort 5 was determined safe for Phase II studies. Thromboembolic events were observed in 9% of patients (no prophylactic coumadin was used). Plasma concentrations of EMP and metabolites increased proportionally with dose. In all cohorts, there was a slight decrease in EMP and estramustine plasma concentrations between cycles 1 and 2. Although not significant, higher levels of estromustine at cycle 2 were observed in comparison to cycle 1. Decreased clearance of paclitaxel leading to higher than expected paclitaxel plasma concentrations was observed during the first cycle of therapy. Paclitaxel plasma concentrations were lower during cycle 2. In 17 patients with androgen-independent disease, 59% had/=50% posttherapy decline in PSA and 22% showed measurable disease regression.The regimen of weekly i.v. EMP in combination with paclitaxel and carboplatin can be safely administered with hepatic toxicity being transient and reversible. Pharmacokinetic results suggest that EMP competitively inhibits the biotransformation of paclitaxel after the first administration. This effect is counterbalanced, after repeated administrations, by a possible induction of the metabolic system caused by EMP. Phase II testing is ongoing to evaluate the efficacy of this combination.

PURPOSE: To determine the dose-limiting toxicities, maximum-tolerated dose, and pharmacokinetics of intravenous estramustine phosphate (IV EMP). PATIENTS AND METHODS: A total of 31 patients with hormone-refractory prostate cancer received IV EMP as a 30- to 90-minute infusion weekly (n = 28) or for 3 consecutive days followed by a single weekly dose (n = 3). IV EMP dose was escalated from 500 to 3,000 mg/m2. Pharmacokinetics of EMP and the metabolites estramustine (EaM), estromustine (EoM), estradiol, and estrone were assessed after weeks 1 and 4 of treatment. RESULTS: The initial IV EMP infusion caused perineal discomfort that was ameliorated by lengthening the infusion time. Other common toxicities were grade 1 to 2 hepatotoxicity, nausea or vomiting, and fatigue or malaise. Lower-extremity thrombosis occurred in one patient, and two others developed upper-extremity thrombosis associated with venous infusion catheters. Dose-limiting fatigue and hypotension occurred at 3,000 mg/m2, and cumulative fatigue developed after multiple cycles at 2,500 mg/m2. Mean EMP clearance, estimated steady-state volume of distribution, and elimination half-life were 3.7 L/h, 10.6 L, and 3.7 hours, respectively. Variability of EMP clearance was 21%, and variation in area under the curve per dose for the metabolites was 28% to 36%. Elimination half-lives of EoM and EaM were 110 hours and 64 hours, and peak plasma concentrations of these active metabolites exceeded 10 μmol/L after IV EMP doses ≥ 2,000 mg/m2. CONCLUSION: High-dose IV EMP can be administered safely as a weekly short infusion to patients with HRPC. High peak concentrations of active metabolites after IV EMP may provide an advantage over oral EMP in antimicrotubule drug combinations.

Objectives To define guidelines for the assessment of treatment in patients with hormone-refractory prostate cancer (HRPC). Methods In the light of modern research, and taking new treatment options into account, the Committee essays to specify different categories of patients entering clinical trials, and to define response criteria and those endpoints that are relevant in phase III studies and in short-term follow-up. Results HRPC comprises a range of disease states with varying responsiveness to therapy and length of survival. Patients with progression as evaluated by increasing prostate-specific antigen (PSA) values alone have a more favorable prognosis than those presenting with increasing tumor spread. In the assessment of these patients, the modes of previous therapy and the kind of tumor progression must be taken into account. The benefit of treatment of HRPC is often modest. While duration of survival remains the main and ultimate endpoint, the means of measuring short-term responsiveness to therapy are limited. A minority of patients have measurable tumor lesions. Decrease of PSA or other biochemical tumor markers may indicate depression of the tumor activity, but is not always associated with prolongation of survival. A variety of new treatments in HRPC are being investigated. They affect measurable tumor parameters in different ways. Conclusions When a new agent is to be tested, it is important to measure all possible parameters before deciding which particular ones are appropriate for future investigations of this agent. In symptomatic patients, evaluation of subjective parameters, for example, relief of pain or improvement of performance status, is often the most reliable measure of treatment effect. However, these parameters should be clearly defined.

The interaction of the antimitotic agent estramustine with bovine microtubule proteins and purified tubulin was investigated. Direct photoaffinity labeling of microtubule protein with [14C]estramustine resulted in the labeling of both alpha- and beta-tubulin, and this was inhibited with unlabeled estramustine in a dose-dependent manner. [14C]Estramustine was incorporated into both the soluble and polymerized forms of tubulin. The affinity constant for estramustine binding to tubulin was determined by equilibrium dialysis to be 23 +/- 5 mM. Estramustine did not affect [3H]vinblastine binding, and vinblastine had no effect on direct labeling with [14C]estramustine. Both rhizoxin and paclitaxel decreased the covalent labeling of tubulin with [14C]estramustine in a dose-dependent fashion and were noncompetitive inhibitors of the binding of estramustine to tubulin. The binding of colchicine to tubulin was not inhibited by estramustine as detected by fluorescence and DEAE filter assays. The estramustine binding site on tubulin is therefore distinct from that of colchicine and vinblastine and may at least partially overlap with the binding site for paclitaxel. In both bovine brain microtubules and cytoskeletal proteins from human prostatic carcinoma cells, the incorporation of [14C]estramustine into the beta III isotype of tubulin was found to occur with a reduced efficiency compared to that of the other beta-tubulin isotypes and alpha-tubulin. Since this isotype is overexpressed in estramustine resistant human prostate carcinoma cells, these results indicate that beta III-tubulin may play a role in the response to the effects of estramustine.

Tauromustine (TCNU) is an exploratory drug that has demonstrated activity in various solid tumors. We examined chromosome aberrations and plasma levels of the drug in two groups of patients receiving either a single dose of 130 mg/m2 or 40 mg/m2 on 3 consecutive days. Peak plasma concentrations (mean +/- SD) were obtained at a similar time after both treatments, i.e., at 38 +/- 25, 32 +/- 24, 28 +/- 14, and 40 +/- 26 min after administration of 130 mg/m2 on day 1 and after that of 40 mg/m2 on days 1, 2, and 3, respectively. In addition, the cumulative area under the curve (AUC value) determined after administration of 40 mg/m2 x 3 was similar to that noted after treatment with a single dose of 130 mg/m2, i.e., 180 and 179 micrograms min ml-1, respectively. Both treatments induced chromosome aberrations (CAs) in peripheral blood lymphocytes. A dose-dependent increase in the number of CAs was found, with 40 mg/m2 producing 5.5% CAs and 130 mg/m2 yielding 20.9% CAs at 24 h after treatment. In addition, although the drug concentration declined to a level under the detection limit between the daily treatments, drug-induced chromosome damage was cumulative, with the 90-min values increasing from 4.8% on day 1 to 14.3% CAs on day 3. In individual patients, no correlation was found between CAs and kinetic parameters; however, the total mean CA yield was in agreement with the total drug exposure (CAs, 14.3% and 14.6%, AUC 180 +/- 62.8 and 179 +/- 115 micrograms min ml-1, respectively.

Linomide, a quinoline-3-carboxamide, has growth-inhibitory effects against a series of Dunning R-3327 rat prostatic cancers in vivo [Ichikawa et al.: Cancer Res 52:3022–3028, 1992]. In addition, we have demonstrated that daily linomide treatment can inhibit angiogenic responses in nontumor-bearing rats and reduce tumor blood flow in tumor-bearing rats [Vukanovic et al.: Cancer Res 53:1833, 1993]. In the present study we have demonstrated that the reduced tumor blood flow is due to linomide's ability to inhibit tumor angiogenesis, as documented by decreased number of blood vessels in prostatic carcinomas growing in rats treated daily with linomide. Due to linomide's ability to inhibit tumor angiogenesis, and since tumor angiogenesis is required not only for the growth of the primary cancer but also for its ability to metastasize, the effect of linomide on metastasis was directly tested using a quantitation metastasis assay. These in vivo experiments demonstrated that daily linomide treatment decreased by 3-fold the extent of dissemination of cancer cells to the lungs. To test if this antimetastatic response is due to direct effects of linomide on the metastatic cells themselves as well as an induced effect upon inhibition of tumor angiogenesis, additional studies were performed. These studies demonstrated that linomide is not converted in vivo to metabolite(s) which are directly cytotoxic or cytostatic to the prostatic cancer cells themselves. These studies also demonstrated that linomide does not decrease the attachment, migration, or invasive abilities of metastatic cancer cells. These results suggest that the major mechanism for the antitumor and antimetastatic effects of linomide is via its inhibition of tumor angiogenesis. Additional studies have demonstrated that in vivo linomide treatment results in the apoptotic death of thymocytes. This cytotoxic effect is not required for linomide's antitumor effect, nor is it due to elevated plasma levels of glucocorticoid.

Nor-nitrogen mustard is an alkylating agent used in kilogram quantities in the production of various cytostatics. It has been shown to be mutagenic in vitro, and therefore must be regarded as a health hazard. We have studied the environment and blood of a small group of individuals working with nor- nitrogen mustard. New chemical methods for measuring surface contamination were used to make comparisons with ambient air levels of nor-nitrogen mustard. Surprisingly high levels of surface contamination were found even after decontamination, which has led to new decontamination routines and the establishment of wipe test limits of 0.5 μg nor-nitrogen mustard/dm 2. The wipe test has proved effective in following the day-to-day routine handling of nor- nitrogen mustard. Blood samples were taken before, during, and after production runs and analyzed for the frequency of chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and somatic (HPRT) mutations. No significant differences in chromosome aberrations, SCEs, or somatic mutations were found in the peripheral blood from production workers before, during, or after handling of nor-nitrogen mustard. Nor was any difference found between the production workers and a local control group. However, the local controls' chromosome and HPRT frequencies appeared to be somewhat high, demonstrating the problems involved in these types of studies, in which the group sizes are small.

Tauromustine (TCNU) a new taurine-based nitrosourea in phase III clinical trials against colon cancer, has been tested and compared with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (CCNU) for mutagenic potential in Salmonella typhimurium TA98 and TA100 and, for its ability to cause chromosome aberrations and sister chromatid exchanges (SCE) in human lymphocytes. A dose dependent increase in the number of mutations in S. typhimurium TA100 but not in TA98 was found from 1.6 to 1000 micrograms/plate for both TCNU and CCNU. In addition, in the presence of Aroclor activated liver microsomes, CCNU caused a further increase in the mutation frequency which was not found for TCNU. A dose dependent increase in the chromosome aberration rate in human lymphocytes was induced by both TCNU and CCNU at concentrations ranging from 3.75 to 30 micrograms/ml after 24 h treatment during the last cell cycle. When lymphocytes were treated in G0 both TCNU and CCNU induced a higher frequency of aberrations at 15 and 30 micrograms/ml than during the last cell cycle and, unexpectedly, chromosome-type, i.e. dicentrics appeared. The difference in the frequency of aberrations in these two phases may be related to the different levels of O6-methyl guanine transferase present in resting contra cycling lymphocytes. TCNU at 0.8-8 micrograms/ml also induced a dose dependent increase in the number of SCE in human lymphocytes. Neither the number of chromosomal aberrations nor the number of SCE were affected by the addition of 1000 micrograms/ml taurine to the culture medium.

Expression of estramustine-binding protein (EMBP) was determined in eight variants of the Dunning R3327 rat prostate adenocarcinoma. This secretory protein was previously isolated from the normal rat prostate and binds estramustine and estromustine, the metabolites exerting anti-mitotic and anti-proliferative activity of the anticancer agent estramustine phosphate (EstracytR). EMBP was found in relatively high amounts only in the androgen-responsive G and H tumors from intact hosts, whereas low (AT-1, HI-F) or nondetectable levels (HI-S, AT-3, MAT-LyLu, MAT-Lu) were obtained in the androgen-independent tumors. Castration decreased EMBP expression in both G and H tumors, as did estradiol and estramustine when given separately to intact rats. Supplementation of castrates with exogenous androgen stimulated EMBP expression above the pre-castration level and was further enhanced when combined with estradiol and in particular estramustine. Partial physicochemical characterization of EMBP in G and H tumors was performed by using ligand-based and immunoblotting techniques. [3H]Estromustine was bound to cytosols and salt extracts from tumors with the same affinity and displayed similar surface-charge distribution, sedimentation behavior, and subunit composition as found for ventral and dorsolateral prostatic tissue from tumor-bearing rats. This study indicates that the synthesis of EMBP is under androgenic control and that its expression may be correlated to androgen responsiveness, metastatic potential, and androgen receptor content but not to growth rate and morphology of the tumors. EMBP may therefore provide a mechanism for concentration of cytotoxic activity at the target site in EMBP-positive tumors and help in the evaluation of antitumor effects obtained when administering estramustine.

Calcium and calmodulin play significant roles in DNA synthesis and cell proliferation. In this work the effects of verapamil, trifluoperazine, and tamoxifen on 45Ca uptake and cell growth in human prostatic tumor cells (DU 145) and human fibroblast cells (1 BR) were studied. Although the maximum proliferation was achieved at a concentration of around 2 mM CaCl2 in both DU 145 and 1 BR, growth of DU 145 cells was considerably greater than 1 BR at all calcium concentrations (0.1-4 mM). Calcium uptake experiments, using 45Ca, revealed that the unstimulated 45Ca uptake in 1 BR fibroblasts was 4-5 times higher than in DU 145 cancer cells. Depolarization with high extracellular K caused a 2-3-fold increase in 45Ca influx in 1 BR but only 25-55% increase in DU 145 cells. Verapamil caused a significant inhibition of cell growth with an IC50 value of 55 microM. Verapamil paradoxically increased 45Ca uptake in both unstimulated and K-stimulated DU 145 cells. Whereas unstimulated 45Ca uptake could be blocked by very low concentrations of lathanum (10 microM), much higher concentrations (1-10 mM) were required to completely block uptake in K-depolarized cells. Both trifluoperazine and tamoxifen also inhibited cell proliferation with an IC50 concentration of approximately 5 microM. These drugs, had, however, no effect on 45Ca uptake either in unstimulated or depolarized cells. The results suggest that voltage-gated calcium channels exist in both DU 145 cancer cells and fibroblasts. However, verapamil, in contrast to 1 BR, failed to block these channels in DU 145 cells. The mechanism of antiproliferative action of verapamil may be related to the observed, although paradoxical, increase in cellular calcium. The effect of trifluoperazine and tamoxifen does not involve changes in transmembrane calcium movements but could be mediated by their inhibition of calmodulin-mediated reactions within the cell.

The binding characteristics of 3H-quinuclidinyl benzilate (QNB) to muscarinic sites in isolated plasma membrane fractions from R-3327 Dunning tumors (H and AT-1 sublines); ventral, dorsolateral prostate; and urinary bladder of the rat were studied. QNB binding to all preparations, except from AT-1 tumors, was specific, saturable, and of high affinity. The AT-1 tumors completely lacked specific QNB binding. The muscarinic receptor density in H tumors was twofold and twentyfold higher than that in the ventral prostate and dorsolateral prostate respectively. The receptor density in the urinary bladder was approximately twofold higher than that in H tumors. The Kd values in H tumors and ventral prostate were very similar and significantly higher than that in dorsolateral prostate or the urinary bladder. QNB binding in H tumors was strongly inhibited by classical muscarinic receptor antagonists atropine and scopolamine, but poorly by the agonists carbacholine and pilocarpine. In contrast to scopolamine or atropine, inhibition by pirenzepine and AF-DX116 was relatively low. These data indicate that the muscarinic receptor in Dunning H tumors is of M3 type.

To further clarify the mode of action of estramustine, the influence on macromolecular synthesis in the human prostatic tumour cell line 1013L was investigated. Cell treatment with estramustine, nor-nitrogen mustard and tauromustine, followed by radioactive nucleotide and leucine incorporations, as a measure of RNA, DNA and protein labelling, were carried out. The initial effect of estramustine clearly differed from that obtained after treatment with nor-nitrogen mustard and tauromustine. No inhibition of DNA synthesis was found whereas an inhibition of overall RNA synthesis was predominant. Adaption of an established RNA separation method was used in an indepth study of RNA labelling after estramustine treatment. An inhibition of 29S, 18S and 4-7S RNA was found after estramustine treatment, indicating disturbances in either RNA processing or RNA transport. The lack of 45S RNA labelling additionally indicates pre-ribosomal inhibition.

Using cultured HeLa S3 cells an ID50 of 2.5 micrograms/ml was found after a twenty-four-hour incubation with estradiol-17 beta- 3N -bis-(2-chloroethyl) carbamate (estramustine). Similar ID90 values were found in two Walker 256 rat carcinoma cell lines which were either sensitive or resistant to nitrogen mustards. Alkaline elution methodology revealed the complete absence of DNA strand breaks or cross-links in cells receiving up to 10 micrograms/ml estramustine for twenty-four hours. Nuclear uptake was 1.34 per cent of the available drug, one third of which was hydrophobically associated with the protein/phospholipid components of the nuclear matrix. In the human prostatic cell lines DU145 and PC3 , estramustine caused a drastic dose-dependent increase in the mitotic index. This increase resulted from an arrest of cells in metaphase, with highly contracted disoriented chromosomes present. Rapid reverse of the arrest on removal of drug resulted in cell death. Neither nor-nitrogen mustard nor estradiol demonstrated antimitotic properties. The lack of macromolecular alkylation together with the observed antimitotic effects predict a mechanism of action for estramustine which is distinct from either of its constituent components.

Nitrogen mustard and nor-nitrogen mustard were investigated in Salmonella typhimurium and human lymphocytes for genotoxicity. Point mutations were assessed using the plate test, the conventional spot test and a method in which the substances were not in direct contact with the microorganisms. Both compounds were active, but nor-nitrogen mustard was far more potent than nitrogen mustard in all bacterial systems. Cytogenetic experiments with human lymphocytes revealed that both compounds induced a dose dependent increase in chromosomal aberrations and sister chromatid exchanges, but that nitrogen mustard was 10 times more potent than nor-nitrogen mustard. Thus, the spectrum of activity for nor-nitrogen mustard and nitrogen mustard differ. Nor-nitrogen mustard was more effective in inducing point mutation damage than nitrogen mustard and a reversal of potency was found for cytogenetic damage. These results indicate that although these two substances induce the same type of DNA lesions the amounts of the different DNA adducts vary.

The early effects of X-rays, vincristine, cyclophosphamide, quinacrine dihydrochloride, cycloheximide, actinomycin D and hydroxyurea on the induction of micronuclei in mouse bone-marrow erythrocytes were studied. A significant increase in the incidence of micronuclei in polychromatic erythrocytes was seen as early as 5 h after a single treatment with vincristine, 6 h after treatment with X-rays and 10 h after treatment with cyclophosphamide. The cell kinetics of the mouse erythropoietic system described by Cole et al. (1981) can be modified to fit these results. According to this revised model, the final mitosis takes place only 5 h before the expulsion of the nucleus.

Chromosomal analysis was carried out on the peripheral lymphocytes of 12 patients receiving metronidazole 200 mg t.i.d. for 7 days for Trichomoniasis vaginalis infections. Samples were taken before, during and 3 weeks after completion of treatment, each patient thus acting as her own control. No increase in the chromosomal aberration frequency was found for any aberration type, either for the individual or for the group means. Thus metronidazole during short-term treatment was not found to exhibit any clastogenic effect on human lymphocytes in vivo.

The clastogenic effect of mitomycin C (MC) was determined in two normal fibroblast cell lines and two xeroderma pigmentosum (XP) cell lines, a variant and a group A excision-deficient line. The group A xeroderma cell line was substantially more sensitive to MC than either the XP variant or the normal human cells. On caffeine post-treatment potentiation of the MC-induced aberration frequency occurred in all the cell lines. The XP varian cell line exhibited a distinctly higher sensitivity to caffeine than the classical XP or the normal human cell lines.

The potentiating effect of 5times10-3M hydroxyurea in G2-prophase on chemically induced chromosomal aberrations has been studied in Chinese hamster cells. Hydroxyurea, when given during the last 2 h before fixation, enhanced the frequency of aberrations induced by both S-dependent (thiotepa) and S-independent (bleomycin) agents. The most striking effect was seen on the frequencies of gaps and chromatid breaks but an increase was also found in the number of iso-chromatid breaks of the non-union type. The frequencies of exchanges and iso-chromatid breaks of the sister-union type were comparatively little affected. The results indicate that hydroxyurea inhibits a repair system in G2-prophase involved in the formation of chromosomal aberrations in mammalian cells.

Chromosome analysis was carried out on peripheral blood lymphocytes from 12 women at 0, 1, and 4 weeks. Two women were also consecutively sampled for 2 years. The mean rate for chromatid breaks was 3.0% range 0–13 and for gaps 8.7% range 2–21.

A novel nitrosourea, 1-(2-chloroethyl)-3-[2-(dimethylaminosulfonyl) ethyl]-1-nitrosourea (TCNU) tauromustine, has been investigated in a broad anti-tumour screen and, in depth toxicology and initial pharmacokinetics carried out. TCNU and its two metabolites were found to exhibit equal or better oral efficacy than that of BCNU, CCNU, MeCCNU or chorozotocin against L1210 leukemia, Walker mammary carcinoma, Lewis Lung, Harding Passey melanoma and colon carcinoma C26. The toxicological profile of TCNU after acute and 3 months treatment was similar in mice and rats to that of CCNU, with the exception that, TCNU did not cause the chronic liver disturbances found for CCNU. In dogs treated for 6 weeks with TCNU leucopenia and thrombocytopenia were the major side effects. Parent TCNU was found in all dogs. The absorption was fast, the maximum level being reach after 25 mins and the mean absorption time was 22 mins. The mean half life was 16.1 mins after intravenous and 17.4 after oral administration. The combination of these factors make TCNU an interesting clinical candidate.

Drug induced DNA alterations have for several years been accepted as ultimately leading to cell death. Less attention has by comparison been given to drug interactions with nuclear proteins. The nuclear protein matrix has recently, however, come into focus as an extremely important structure with regard to replication sites, hormone receptors, nuclear shape etc. (see Tew, this volume). Thus, drug interaction with the nuclear matrix could lead to severe disturbances in cell growth.

A Walker 256 breast carcinoma cell line (WR) exhibiting a greater than 20-fold resistance to alkylating agents has been selected from a parent cell line (WS). Karyotypic heterogeneity was apparent, with a number of differences evident between WR and WS cells. The modal chromosome number for WS is 62; for WR, 54; double minutes were found only in WR, whereas spontaneous chromosomal aberrations were present in approx. 40% of the WS cells. No similar aberrations were observed in WR. Using SDS-gel electrophoresis and subsequent silver staining, differences in the profile of nuclear matrix proteins in WR and WS were observed. A diffuse band at approx. 70 kD in the WS was absent in WR cells. This protein was phosphorylated, together with a number of the other major matrix polypeptides. Levels of phosphorylated matrix proteins were approximately equivalent in both WR and WS cell lines, but matrix protein phosphorylation levels were approx. 2-fold higher than corresponding values for bulk nuclear proteins. Selective pressure of drug exposure has resulted in enhanced genetic stability in WR cells and observed karyotype differences are accompanied by modifications in the structural proteins of the nuclear matrix. Whether the observed differences are the cause or result of drug resistance remains to be established.

The effect of estramustine [estradiol 3-N-bis(2-chloroethyl)carbamate] on the human prostatic tumor cell line 1013L was investigated. Cell proliferation experiments revealed that estramustine cytotoxicity varied during the different phases of cell growth. Maximum cell killing was found in early log phase, but cell death also occurred in the stationary phase. Mitotic arrest was found at cytotoxic concentrations throughout the log phase. Subcellular distribution studies showed that the cellular uptake of estramustine increased throughout the log phase and remained steady during the stationary phase. Nuclear uptake in contrast was similar in all phases, whereas a preferential binding to the nuclear protein matrix was found to increase throughout the log phase and even during the stationary phase of growth. This implicates the nuclear protein matrix as a target for estramustine cytotoxicity.

Two human prostate carcinoma cell lines are easily distinguished by C-banding. DU 145 has one metacentric chromosome with a large centromeric band, 1 metacentric and 2 acrocentric chromosomes with interstitial C-bands. The Y chromosome was present in 48% and 42% of the cells at passages 83 and 106, respectively, whereas in 1013L only one metacentric chromosome contained distinct extra C bands--one at the centromere and one interstitially. The Y chromosome was present in 72% of the cells at passage 15 and in 20% at passage 22, but by passage 44 it had disappeared. Karyotype analysis using G-banding revealed that DU 145 had retained several of its original markers, and demonstrated multiple marker chromosomes in 1013L.

Prednimustine and chlorambucil induce dose- and time-dependent cell death in V79 Chinese hamster cells in vitro. Prednimustine was found to be 3-4 times more potent than either chlorambucil or an equimolar mixture of its components chlorambucil and prednisolone after 24 h treatment. Prednimustine was hydrolyzed to prednisolone and chlorambucil in the system, and the concentration of prednimustine was reduced by one half within 15 h. Prednisolone was not further metabolized, but chlorambucil was rapidly inactivated by dechlorination, the half-life being 2.5 h. No dechlorinated prednimustine was formed during the experiments. The higher stability of prednimustine than chlorambucil is probably due to protective binding to different serum proteins from those that bind chlorambucil. Substitution of fetal calf serum by human serum albumin revealed that hydrolysis of prednimustine is catalyzed by esterases present in the serum. In similar substitution experiments cell survival studies indicated that prednimustine itself was not cytotoxic. Rather, cytotoxicity was found to correlate with hydrolysis to chlorambucil. Thus, it appears that the prolonged availability of chlorambucil is responsible for the increased potency of prednimustine in this system.

In Vicia faba, Allium proliferum and Nigella damascena root-tips, Cricetulus griseus and Potorous tridactylis cells an enhancement of the chromosome aberration frequency induced by Mitomycin C and Thiotepa was obtained in all materials after long post-treatments with 5 times 10-4 M caffeine, at various fixation times. In Vicia faba it was demonstrated that the most sensitive period for caffeine potentiation was 9–12 h after the beginning of the inducer treatment. This period corresponded to the middle-late S phase after Mitomycin C treatment in Vicia faba root-tips. Synergism was also evident after maleic hydrazide plus caffeine exposure in Vicia faba and Allium proliferum root-tips. The threshold value for caffeine potentiation after Mitomycin C was found to be the same for both Vicia faba root-tips and Chinese hamster cells (Cricetulus griseus), that is between 2–3 times 10-4 M. In all the materials, except Allium proliferum, the potentiation factor was higher for Thiotepa than Mitomycin C.

Tumor cells from of the Dunning R-3327 PAP tumor were grown in vivo in the flank region of male Copenhagen rats for 4 months. Untreated control animals, castrated animals, and untreated or castrated animals supplemented with testosterone and estramustine (alone or in combination) bearing tumors were used for immunocytochemical studies of the tumor cells. Antibodies against the following secretory proteins were applied to paraffin sections of formalin-fixed tissue: anti-SVS 11, anti-PBP, anti-transglutaminase, anti-acid phosphatase (isoenzymes, isoelectric points [PI] 5. 6, 7, 1, 8, 0), non-secretory proteins comprised antiglucocorticoid-receptor, and antibodies against extracellular matrix proteins and intermediate filaments. A differential expression of marker proteins subsequent to the various treatments was observed immunohistochemically. Castration induced a loss of secretory activity, an increase in keratin-immunoreactive cells, and regressive activity in the secretory cells. Immunoreactivity of the glucocorticoid receptor in the nuclei of the basal cells was also decreased. Testosterone substitution only partially restored secretory activity of tumor cells in castrated animals. In experiments where estramustine had been administered to normal or castrated animals, metaplastic transformation of the epithelium, focal reduction, or increase of secretory and/or regressive activity and persistence of glucocorticoid receptor-like imniunoreactivity was observed. The findings indicate that different hormonal situations provide conditions for the development of rather heterogeneous reaction patterns of different tumor cells, allowing partial regression of individual clones of tumor cells along with stimulation of others.

LS 1727, a nitrosocarbamate of 19-nortestosterone, was active against lymphoid, antimetabolite-sensitive, cell lines in vitro especially L1210 and Ehrlich ascites tumour lines. It was less effective against alkylating agent-sensitive lines such as the Walker 256 carcinosarcoma. These results suggested a possible antimetabolite mode of action but LS 1727 had no effect on deoxy- or ribonucleotide pool sizes in L1210 cells. Studies on macromolecular synthesis showed an early inhibition of DNA synthesis whilst RNA and protein synthesis continued for 24–48 hours. This was reminiscent of a classic alkylating agent-like effect. In vivo studies with specific alkylating agent-sensitive, or antimetabolite-sensitive tumours showed no antitumour activity although significant inhibition of Ehrlich ascites tumour cell growth was observed at high doses. In vitro cytotoxicity studies showed LS 1727 to be inactivated in the presence of mouse, rat and dog liver supernatants. This would explain the poor antitumour effects in vivo compared with the in vitro observations. Because LS 1727 is a nitrosocarbamate its possible mutagenic activity was investigated. LS 1727 was highly mutagenic but this property was also lost in the presence of a rat liver microsomal fraction. Although an effective cytotoxic agent in vitro LS 1727 is only effective in vivo at toxic doses.

The metabolism of estramustine [estradiol-3-N-bis(2-chloroethyl) carbamate] was investigated in the human prostatic tumour cell line 1013L and the human cervix tumour cell line HeLa S3. Uptake studies revealed that estramustine (EM), and its 17-ketoanalogue estromustine (EoM), differed in their nuclear binding pattern in 1013L cells but not in HeLa cells. Most of the nuclear radioactivity from both EM and EoM was found in the fraction containing the majority of the phospholipids. HPLC studies on EM-treated 1013L cells showed the presence of the oxidized metabolite EoM, in the medium, an enrichment of estradiol and estrone in whole cells and EM and EoM bound to the nuclear protein matrix. Similar studies on the HeLa cell line showed a completely different pattern, no metabolites other than EoM were found in the cell medium and whole cells but several very lipophilic metabolites were found bound to the nuclear protein matrix. On investigation of other tumour cell lines these metabolites were found to be unique to HeLa cells. The results extend our knowledge concerning EM and demonstrate that the cell line 1013L is a relevant model system for studying drugs active against human prostatic tumours.

Estramustine at concentrations ranging from 3–40 × 10−6 m inhibited the cell growth and clonogenic survival of a human prostatic carcinoma cell line (DU 145). This cell line was found to be unresponsive to estradiol and testosterone at concentrations ranging from 10−9 m to 5 × 10−5 m . Metabolism studies with estramustine showed that only a few per cent of the ester linkage was cleaved during the exposure period. This small amount of metabolism could possibly lead to the release of nor-nitrogen mustard, which was however found not to be as inhibitory as estramustine in this cell line. The results indicate that estramustine per se causes the cell death of hormone unresponsive human prostatic carcinoma cells in cell culture.

In growth proliferation experiments on two human prostatic carcinoma cell lines, DU 145 cells were found to be more sensitive to the cytotoxic effect of estramustine and nor-nitrogen mustard than PC-3 cells. Estramustine was, however, much more cytotoxic in both cell lines than nor-nitrogen mustard. Cytogenetic experiments revealed that estramustine produced a drastic increase of the mitotic index in both these cell lines. This increase could be accounted for by the arrest of cells in their first treatment-metaphase. The arrested metaphases exhibited all the characteristics commonly found for stathmokinetic agents such as colchicine and vinca-analogues. No mitotic arrest was found for nor-nitrogen mustard but chromosomal aberrations were found at toxic concentrations. Estradiol exhibited minimal toxicity and caused no mitotic arrest in these cell lines. The mitotic arrest induced by estramustine was found to be reversible on removal of the drug.