Cytotoxic, cell cycle, and chromosomal effects of methylxanthines in human tumor cells treated with alkylating agents.

Human tumor cells, like rodent cells, are sensitive to effects of methylxanthines (MEX) on lethality, cell cycle delays, and chromosome aberrations after DNA damage by anticancer drugs. Enhanced cytotoxicity of alkylating agents was observed when T24 human bladder tumor cells in culture were exposed to nontoxic concentrations of MEX such as caffeine or pentoxifylline. Tumor cell lethality was increased up to 10-fold by either caffeine or pentoxifylline (1 mM) present during the first cell cycle (16-24 h) after exposure to nitrogen mustard (HN2) or thiotepa. Cycloheximide, a protein synthesis inhibitor, abolished the enhanced lethality produced by MEX. In these synchronized human tumor cells further kinetic studies revealed that HN2 (0.5 microM X 1 h) delayed transit through S phase by about 1-2 h, and this delay was prevented by MEX. After completion of S phase, HN2-treated cells were delayed 3-6 h in G2, and MEX also prevented this delay, leading to mitoses at the rate of controls. Chromosome analysis of these mitotic cells revealed dramatic increases in aberrations induced by alkylator + MEX combinations. The greatest number of aberrations was seen in HN2-treated cells exposed briefly to MEX in late S-G2. In contrast, no increased chromosome damage was seen in cells exposed to MEX in mid-S phase. Taken together, our results are consistent with the model that MEX enhance lethality of alkylator-treated human tumor cells by preventing delays in cell cycle transit through G2, leading to chromosome aberrations which are lethal. G2 delays in human tumor cells may provide time for repair processes that are critical for survival after sublethal DNA damage by HN2 or other anticancer alkylating agents.

In vitro evidence that myocardial ischemia resulting from 5-fluorouracil chemotherapy is due to protein kinase C-mediated vasoconstriction of vascular smooth muscle.

5-Fluorouracil (5-FU) is a commonly employed chemotherapeutic agent. Among the various toxicities associated with 5-FU, cardiovascular toxicity, consisting principally of acute myocardial ischemia and/or myocardial infarction, has been reported in up to 8.5% of patients treated with this drug. While 5-FU-induced coronary vasospasm has been considered as a potential basis for such clinical toxicity, this hypothesis remains unsubstantiated by laboratory investigation. Accordingly, the present study was designed to investigate the hypothesis that 5-FU induces reversible vasoconstriction of vascular smooth muscle and to study the cellular mechanisms of such vasomotor alterations. To investigate the effects of 5-FU on the vasoreactivity of vascular smooth muscle, 479 exposures were performed in 105 rings of aorta freshly isolated from 23 New Zealand white rabbits. Vasoconstriction was documented in 20 of 86 (23%) rings exposed to 5-FU at 7 x 10(-5) M, 45 of 83 (54%) rings exposed to 5-FU at 7 x 10(-4) M, and 41 of 49 (84%) rings exposed to 5-FU at 7 x 10(-3) M. In each case, 5-FU-induced vasoconstriction was endothelium independent. Pretreatment of rings with 10(-9) M staurosporine, a protein kinase C (PK-C) inhibitor, reduced 5-FU-induced vasoconstriction from 25.0 +/- 6.5 to 2.5 +/- 1.7 mg; staurosporine at a concentration of 10(-8) M abolished 5-FU-induced vasoconstriction. Pretreatment of rings with 10(-7) M phorbol-12,13-dibutyrate, an activator of PK-C, increased the magnitude of 5-FU-induced vasoconstriction 23-fold, from 49.7 +/- 11.1 mg before to 1163.6 +/- 276.4 mg after phorbol-12,13-dibutyrate (P = 0.0002). Neomycin, an inhibitor of phosphoinositide turnover, did not alter the magnitude of 5-FU-induced vasoconstriction. Membrane receptor blockers, including the alpha-adrenergic receptor blocker phentolamine, the beta-adrenergic receptor blocker propranolol, the H1 receptor inhibitor diphenhydramine, the H2 receptor inhibitor cimetidine, the Ca2+ channel blockers verapamil and diltiazem, and the cyclooxygenase inhibitor indomethacin all failed to alter the magnitude of 5-FU-induced vasoconstriction. Furthermore, the 5-FU-related compounds uracil and floxuridine did not produce vasoconstriction. Finally, 5-FU-induced vasoconstriction was abolished by nitroglycerin. These results indicate that (a) 5-FU causes direct, endothelium-independent vasoconstriction of vascular smooth muscle in vitro, (b) this vasomotor response involves activation of PK-C, and (c) this response is independent of vasoactive cell membrane receptors, phosphoinositide turnover, or activation of the cyclooxygenase pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclin E, a potential prognostic marker for breast cancer.

A fundamental cause of cancer is changed properties of genetic material, which may deregulate normal development of the tissue or provide selective growth advantage to the tumor cell. This deregulation of cell proliferation results from altered production of a handful of proteins that play key roles in progression through the eukaryotic cell cycle. Some of these proteins include tumor suppressor genes or oncogenes. However, no one general change or alteration of a critical gene has yet been found in all cancers. Using surgical material obtained from patients with various malignancies, we show that breast cancers and other solid tumors, as well as malignant lymphocytes from patients with lymphatic leukemia, show severe quantitative and qualitative alterations in cyclin E protein production independent of the S-phase fraction of the samples. Hence, these alterations represent a true difference between normal versus tumor tissue. In addition, in breast cancer, the alterations in cyclin E expression become progressively worse with increasing stage and grade of the tumor, suggesting its potential use as a new prognostic marker.

Rapid growth of human cancer cells in a mouse model with fibrin clot subrenal capsule assay.

Rapid in vivo growth of cultured human cancer or leukemia cells was achieved by implantation into the subrenal capsule of mice. A solid structure, necessary for accurate implantation and measurement of tumor growth in this model, was provided by stepwise addition of fibrinogen and thrombin to the tumor cells, leading to rapid enzymatic formation of a solid tumor-fibrin matrix. Human leukemia and epithelial cancers increased in volume between 6- and 40-fold when measured 6-10 days after implantation into normal or immunosuppressed mice. Immunosuppression of host CD-1 mice was achieved by cyclosporine given daily after tumor implantation, cyclophosphamide given preimplantation combined with cyclosporine, or whole-body irradiation given preimplantation. Confirming the validity of tumor measurements, tumor histology in the immunosuppressed mice revealed cell proliferation, invasion, and neovascularization. Similarly, no artifactual measurement of tumor growth was observed by nonviable cancer cells, implanted after in vitro exposure to a known cytotoxic concentration of thiotepa. This model provides an economical, short-term technique for the in vivo study of human tumor growth, for the evaluation of new cancer therapies, and for in vitro - in vivo drug activity correlations in specific types of human cancer or leukemia cell lines.

In vivo and in vitro enhanced antitumor effects by pentoxifylline in human cancer cells treated with thiotepa.

Methylxanthines enhance lethality of alkylating agents in human cancer cells, a phenomenon attributed to the prevention of DNA repair. Pentoxifylline is a nontoxic methylxanthine, used clinically for claudication. Using human cancer cells in culture or in a mouse xenograft model, we studied combination treatments with alkylating agents and pentoxifylline or other methylxanthines. With human bladder cancer cells in culture, cytotoxicity of thiotepa was increased up to 10-fold (P less than 0.01) by posttreatment with pentoxifylline, with a major clinical metabolite of pentoxifylline, or with caffeine; the pentoxifylline concentrations required (0.4-1.0 mM) are clinically achievable in the bladder after nontoxic p.o. doses. With human bladder or breast cancer xenografts in a modified subrenal capsule assay, enhancement of thiotepa was also observed by in vivo posttreatment with pentoxifylline. In contrast, these combinations produced no increased toxicity to normal tissues in these animals, measured by weight, lethality, or histological changes of the normal bladder urothelium. These results provide evidence for a novel approach to improve the therapeutic index of thiotepa and other alkylators, used for topical therapy of bladder cancer and, possibly, systemic therapy of other malignancies.

Analysis of c-erbB-2 expression in breast carcinomas with clinical follow-up.

Various monoclonal antibodies reactive with protooncogene products or tumor-associated antigens have been utilized to investigate breast carcinoma biology or antigen expression with potential prognostic relevance. Murine monoclonal antibody TA1, generated by immunization of BALB/c mice with whole c-erbB-2 (neu) transformed NIH/3T3 cells, recognizes the extracellular domain of the c-erbB-2 protein and binds a Mr 185,000 protein by immunoprecipitation. Using avidin-biotin-peroxidase techniques and monoclonal antibody TA1, 313 archival primary adenocarcinomas of the breast were evaluated for c-erbB-2 overexpression; 290 of these were used for multiparametric statistical analysis. Historical, clinical (age, laterality), histological (nuclear grade, tumor size, lymph node status, lymphatic or blood invasion), and hormone receptor data as well as clinical outcome (minimal follow-up, 6 years; median follow-up, 8.5 years) were compared to TA1 staining. For these 290 patients Cox regression multivariate analysis showed the strongest correlation between lymph node status or estrogen receptor status and overall survival (P = 0.0001 and 0.049, respectively). TA1 staining did not significantly correlate with survival (P = 0.395). However, univariate analysis of certain patient subpopulations showed a significant correlation if the examined tumors were subdivided into negative or focally reactive and those with greater than or equal to 40% cellular reactivity. For T3, T4 patients, strong TA1 immunoreactivity correlated with a shortened disease-free survival (log rank P = 0.0018; Wilcoxon p = 0.0078) and overall survival (log rank P = 0.0002; Wilcoxon P = 0.0013). For these patients the overall survival at 6 years was markedly different between the strongly reactive tumors (0%) and the negative to weakly reactive tumors (55%). In lymph node-positive patients a trend between high TA1 reactivity and a worse overall survival was also noted (log rank P = 0.128; Wilcoxon P = 0.054), with a 6-year survival of 42% in the strongly reactive tumors (n = 16) and 65% in the negative to weakly reactive carcinomas (n = 105). No correlation between TA1 immunoreactivity and other historical, clinical, and histological features were noted. c-erbB-2 overexpression as measured by immunohistochemical techniques, therefore, may have clinical significance in certain patient subpopulations.

Implantation of human meningiomas into the subrenal capsule of the nude mouse. A model for studies of tumor growth.

To develop a reproducible in vivo model for the growth of human meningiomas, meningiomas from 16 patients were implanted into the subrenal capsule of the nude mouse. In eight experiments solid tumor implants taken directly from surgical specimens were used, in four experiments the implants were made from early-passage monolayer cell cultures, and in four experiments both techniques were used. Successful tumor growth was observed in 10 (83%) of the 12 solid tumor implants and in six (75%) of the eight implants from cell cultures. The size and neovascularization of these tumors were serially determined over a 3-month period. Tumor doubling occurred in 1 to 3 weeks in all of the solid tumor implant group. In the group of six tumors successfully implanted from cell cultures, three doubled in 1 to 3 weeks and three grew more rapidly, reaching 10 to 20 times their original volume. Neovascularity occurred in the tumors within 3 weeks of implantation. Each of the solid tumor implants had a histological pattern similar to that of the corresponding original specimen. Only three of those implanted from cell cultures were similar to the original tumor; the other three displayed features characteristic of malignant meningioma. These studies suggest that implantation of human meningiomas in the subrenal capsule of the nude mouse is a feasible model that may be useful for evaluating hormonal or genetic modulation of tumor growth and for testing potential treatment regimens.

Performance characteristics of a multimedia system for clinical data management.

This study describes a multimedia, computer-based system developed to record and retrieve clinical images, associated dictated commentaries, and textual and numeric data from patients with cancer or hematologic diseases. Objective performance was analyzed by (1) success rate for data input and retrieval, (2) speed of image retrieval, and (3) quality of stored images. During a 1-year period, 587 diagnostic images were recorded with dictated or typed summaries of pertinent clinical information. Subsequent retrieval of stored data was performed three times (n = 1761) with 100% reliability. The average time for retrieval of random images was 6.4 seconds (SD = 1.7 seconds), including operator time. Image quality was scored by a four-point scale: the average score was 3.58 (SD = 0.5), and all images displayed sufficient quality to represent a wide range of diagnoses. These data demonstrate that multimedia computer technologies can enhance the organization and management of clinical images needed for medical education, clinical trials, and daily care of patients with cancer and hematologic diseases.

Enhanced lethality by methylxanthines in human bladder cancer cells treated with thiotepa.

Cytotoxicity of thiotepa or doxorubicin hydrochloride in human bladder cancer cells was investigated alone and in combination with methylxanthines. Methylxanthines can potentiate cytotoxicity of some DNA-damaging agents used in cancer therapy by preventing DNA repair. However, the potential clinical utility of these drug combinations has not been defined. Nontoxic concentrations of two methylxanthines, theobromine and caffeine, markedly enhanced lethality in T-24 human bladder cancer cells treated with thiotepa. Thiotepa cytotoxicity was increased over 10-fold by continuous treatment with nontoxic concentrations of methylxanthines (0.5 mM), but the major enhancing effect was observed in the 1st 24 hours after thiotepa exposure. By contrast, no such enhanced lethality was observed using higher methylxanthine concentrations and equal or greater cytotoxic treatments with doxorubicin hydrochloride. The amount of enhanced lethality by methylxanthines correlated directly with growth rate of the cells in culture, suggesting that differential enhanced therapeutic effects could be achieved in the treatment of superficial bladder tumors based on the increased proliferative rate of neoplastic bladder cells compared to normal bladder urothelium.

Transplantation of human or rodent tumors into cyclosporine-treated mice: a feasible model for studies of tumor biology and chemotherapy.

Total growth of transplanted human or rodent tumors in the subrenal capsule of mice was much improved by treatment with cyclosporine (CSA, cyclosporin A). Tumor size increased rapidly between days 6 and 12 after implantation, CSA injected on days 1-5 or 2-8 prevented tumor regression. In contrast, immunologic regression occurred after 6 days in absence of the drug. Tumor growth was comparable in CSA-treated mice, athymic nude mice with human tumors, or normal mice with syngeneic rodent tumors. Studies with rodent tumors in syngeneic mice showed that the CSA treatments had no antitumor effect. Inflammatory infiltration was seen on days 6-12 after tumor implantation into control mice. Immunoperoxidase staining showed murine T cells to be prominent in the infiltrate. In contrast, tumors in CSA-treated mice contained minimal inflammatory infiltrate even 12 days after implantation. Allogeneic tumors in CSA-treated mice caused neovascularization, metastases, and local invasion into the kidney. cis-Diamminedichloroplatinum showed highly significant activity against human tumors in CSA-treated mice during the period 6-10 days after tumor implantation but showed no statistically significant antitumor activity 0-6 days after implantation in mice not treated with CSA. We suggest that in CSA-treated mice the subrenal capsule assay for tumor growth provides a rapid, economical model for investigations in vivo of mouse or human tumor biology, for drug screening with a standard tumor, or for determination of optimal treatment of particular human tumors.

Incapacitating autonomic neuropathy precipitated by taxol.

Taxol is one of the more significant advances in cancer chemotherapy in the last 10-15 years with a 30-50% response rate in both breast and ovarian cancers. The toxicity profile of taxol is still evolving as changes in the dosing and scheduling of the drug are instituted. We herein present the first evidence of taxol-induced quantifiable, objective autonomic neuropathy in two patients, one with and one without diabetes. The patients did not have symptoms of autonomic dysfunction prior to taxol therapy. Both developed severe orthostatic hypotension following completion of 24-hr infusional taxol at 170-250 mg/m2 and both improved markedly on fludrocortisone. Other possible etiologies of orthostatic hypotension were ruled out. Noninvasive autonomic tests performed on the first patient included tilt test, Valsalva ratio, heart rate variation with respiration, and change in diastolic blood pressure with hand grip; all were markedly abnormal. Noninvasive autonomic tests should be considered as part of the evaluation of patients on taxol who develop generalized weakness and orthostasis.

Predictive value of the fluorescent cytoprint assay (FCA): a retrospective correlation study of in vitro chemosensitivity and individual responses to chemotherapy.

The potential clinical usefulness of the fluorescent cytoprint assay (FCA) was assessed retrospectively in 73 cancer patients by correlating individual tumor chemosensitivity in vitro with responses to chemotherapy. The data show that the FCA has a sensitivity of 98%, specificity of 81%, and predictive accuracies of 85% and 97% for positive and negative clinical responses, respectively.