Showing papers in "Journal of the National Cancer Institute in 1990"

New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening

Abstract: We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.

Taxol: A Novel Investigational Antimicrotubule Agent

Abstract: Microtubules are among the most strategic subcellular targets of anticancer chemotherapeutics. Despite this fact, new antimicrotubule agents that possess unique mechanisms of cytotoxic action and have broader antineoplastic spectra than the vinca alkaloids have not been introduced over the last several decades--until the recent development of taxol. Unlike classical antimicrotubule agents like colchicine and the vinca alkaloids, which induce depolymerization of microtubules, taxol induces tubulin polymerization and forms extremely stable and nonfunctional microtubules. Taxol has demonstrated broad activity in preclinical screening studies, and antineoplastic activity has been observed in several classically refractory tumors. These tumors include cisplatin-resistant ovarian carcinoma in phase II trials and malignant melanoma and non-small cell lung carcinoma in phase I studies. Taxol's structural complexity has hampered the development of feasible processes for synthesis, and its extreme scarcity has limited the use of a conventional, broad-scale screening approach for evaluation of clinical antitumor activity. However, taxol's unique mechanism of action, its spectrum of preclinical antitumor activity, and tumor responses in early clinical trials have generated renewed interest in pursuing its development.

Comparison of In Vitro Anticancer-Drug-Screening Data Generated With a Tetrazolium Assay Versus a Protein Assay Against a Diverse Panel of Human Tumor Cell Lines

Abstract: The National Cancer Institute (NCI) is implementing a large-scale in vitro drug-screening program that requires a very efficient automated assay of drug effects on tumor cell viability or growth. Many laboratories worldwide have adopted a microculture assay based on metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). However, because of certain technical advantages to use of the protein-binding dye sulforhodamine B (SRB) in a large-scale screening application, a detailed comparison of data generated by each type of assay was undertaken. The MTT and SRB assays were each used to test 197 compounds, on simultaneous days, against up to 38 human tumor cell lines representing seven major tumor categories. On subsequent days, 38 compounds were retested with the SRB assay and 25 compounds were retested with the MTT assay. For each of these three comparisons, we tabulated the differences between the two assays in the ratios of test group values to control values (T/C) for cell survival; calculated correlation coefficients for various T/C ratios; and estimated the bivariate distribution of the values for IC50 (concentration of drug resulting in T/C values of 50%, or 50% growth inhibition) for the two assays. The results indicate that under the experimental conditions used and within the limits of the data analyses, the assays perform similarly. Because the SRB assay has practical advantages for large-scale screening, however, it has been adopted for routine use in the NCI in vitro antitumor screen.

Dietary Factors and Risk of Breast Cancer: Combined Analysis of 12 Case—Control Studies

Abstract: We conducted a combined analysis of the original data to evaluate the consistency of 12 case-control studies of diet and breast cancer. Our analysis shows a consistent, statistically significant, positive association between breast cancer risk and saturated fat intake in postmenopausal women (relative risk for highest vs. lowest quintile, 1.46; P less than .0001). A consistent protective effect for a number of markers of fruit and vegetable intake was demonstrated; vitamin C intake had the most consistent and statistically significant inverse association with breast cancer risk (relative risk for highest vs. lowest quintile, 0.69; P less than .0001). If these dietary associations represent causality, the attributable risk (i.e., the percentage of breast cancers that might be prevented by dietary modification) in the North American population is estimated to be 24% for postmenopausal women and 16% for premenopausal women.

Primary Chemotherapy To Avoid Mastectomy in Tumors With Diameters of Three Centimeters or More

Abstract: In 165 women with breast cancer who were candidates for mastectomy because the largest diameter of the tumor was 3 cm or more, we administered primary chemotherapy in the attempt to substitute conservative for mutilating surgery. We then systematically quantitated tumor reduction by clinical, radiologic, and histopathologic evaluations. Five consecutive groups of 33 patients received cyclophosphamide, methotrexate, and fluorouracil (CMF); fluorouracil, doxorubicin (Adriamycin), and cyclophosphamide (FAC); or fluorouracil, epirubicin, and cyclophosphamide (FEC). The regimens for the five groups were as follows: group 1, three cycles of CMF; group 2, four cycles of CMF; group 3, three cycles of FAC; group 4, four cycles of FAC; and group 5, three cycles of FEC. In response to primary chemotherapy, 157 of the 161 assessable patients showed measurable tumor shrinkage; progressive disease was documented in four. Tumor shrinkage to less than 3 cm was documented in 127 (81%) of the 157 women subjected to surgery, thus allowing a breast-saving procedure, rather than modified radical mastectomy, in these 127 women. Histopathologic complete remission was documented in seven patients. Tumor response was unrelated to age, menopausal status, DNA content (ploidy), [3H]thymidine-labeling index, drug combination used, or number of treatment cycles in excess of three. The degree of response was inversely proportional to the initial tumor size, and the frequency of response was greater in receptor-negative tumors. Severe vomiting and hair loss were less frequent with CMF than with anthracycline-containing regimens, and the frequency of severe leukopenia and thrombocytopenia was minimal. Our results challenge the classical indication for primary mastectomy by showing that use of full-dose primary chemotherapy, sequentially combined with conservative surgery and radiation, can offer an effective and safe alternative to women concerned about the preservation of body integrity.

Curability of tumors bearing herpes thymidine kinase genes transferred by retroviral vectors.

Abstract: Retroviral vectors constructed to contain the herpes simplex virus thymidine kinase (HSV-TK) gene were used for transduction of this gene into murine sarcoma and lymphoma cells to yield sublines susceptible in vitro to the cytotoxicity of ganciclovir, a drug specifically activated by HSV-TK. In vivo, ganciclovir induced complete, durable regressions in most mice bearing transplanted HSV-TK-positive sarcomas; its efficacy against lymphomas was only marginal, possibly because of their greater instability of gene expression. The results imply the potential value of an anticancer strategy entailing the prophylactic use of retroviral vectors to create tissue mosaicism for drug sensitivity.

Dietary Fiber, Vegetables, and Colon Cancer: Critical Review and Meta-analyses of the Epidemiologic Evidence

Abstract: Whether colon cancer risk can be modified by a diet rich in vegetables, grains, and fruit, and, if so, whether the protective factor is dietary fiber or other biologically active components correlated with a high-fiber diet are questions of active research interest. Because studies on diet are susceptible to bias from a number of sources, in this review we evaluated the adequacy of study methodology as well as study results to clarify how much protection, if any, is conferred by a high-fiber diet. The review consisted of an aggregate assessment of the strength of evidence from 37 observational epidemiologic studies as well as meta-analyses of data from 16 of the 23 case-control studies. Both types of analyses revealed that the majority of studies gave support for a protective effect associated with fiber-rich diets; an estimated combined odds ratio (OR) of 0.57 (95% confidence interval = 0.50, 0.64) was obtained when the highest and lowest quantiles of intake were compared. Risk estimates based on vegetable consumption (OR = 0.48) were only slightly more convincing than those based on an estimate of fiber intake (OR = 0.58), but the data do not permit discrimination between effects due to fiber and nonfiber effects due to vegetables.

Endothelial Cell Production of Nitrogen Oxides in Response to Interferon γ in Combination With Tumor Necrosis Factor, Interleukin-1, or Endotoxin

Abstract: Clinical studies using biological response modifiers in cancer therapy have shown that the major dose-limiting toxic effects are hypotension and diffuse microvascular leakage. The cause and pathophysiology of this hypotension remains unknown. Previous experiments have demonstrated that a number of cell types, including endothelial cells, neutrophils, and macrophages, can secrete a potent hypotensive agent--endothelium-derived relaxing factor, which has recently been identified as nitric oxide. In this study, we tested interferon gamma, tumor necrosis factor, interleukin-1, interleukin-2, muramyl dipeptide, and endotoxin for their effects on production of nitrogen oxides by endothelial cells. Interferon gamma, in combination with tumor necrosis factor, interleukin-1 (IL-1), or endotoxin, induced murine brain endothelial cells to secrete nitrites (20-45 microM within 48 hr), which are breakdown products of nitric oxide. Nitrite production was blocked by incubation of endothelial cells in medium without L-arginine, a substrate for nitric-oxide synthase. Accumulation of nitrites was also inhibited by addition of NG-monomethyl-L-arginine (L-NMMA), which acts as a competitive inhibitor of this enzyme. The inhibitory effects of L-NMMA were reversed by addition of excess L-arginine. These results suggest (a) that endothelial cells produce nitric oxide in response to immunomodulators and (b) that endothelial cell-derived nitric oxide plays a role in the development of hypotension in patients treated with tumor necrosis factor or interleukins. Furthermore, administration of substrate analogues such as L-NMMA may favorably alter the toxicity associated with these immunomodulators and result in a higher maximum tolerated dose, with subsequent improvement in the antitumor activity.

Detection and Typing of Human Papillomavirus in Archival Cervical Cancer Specimens by DNA Amplification With Consensus Primers

Abstract: We developed a polymerase chain reaction DNA amplification system using two distinct consensus oligonucleotide primer sets for the improved detection and typing of a broad spectrum of human genital papillomavirus (HPV) sequences, including those of novel viruses. The system incorporates one primer set designed to amplify a highly conserved L1 domain and a second primer set designed to amplify a domain within the E6 gene. We used this system to analyze 48 fixed, paraffin-embedded tissue sections (41 specimens from 33 cervical carcinomas, four normal cervical tissues, and several control tissues) for the presence of HPV DNA. HPV sequences were detected in all carcinoma samples and none of the control samples. Hybridization analyses showed that the results obtained with the two amplification schemes concurred completely. This approach allowed rapid confirmation of typing results and may improve the likelihood of detecting a wide variety of HPV sequences, including those of novel HPVs.

Analysis of Events Associated With Cell Cycle Arrest at G2 Phase and Cell Death Induced by Cisplatin

Abstract: DNA is the accepted target for cisplatin, but recent evidence has shed doubt on DNA synthesis as the critical process. L1210/0 cells incubated for 2 hours with cisplatin progress to the G2 phase of the cell cycle and are arrested there for several days. They then either progress in the cell cycle or die. In cells that eventually die, total transcription, polyadenylated [poly(A)+] RNA synthesis, and protein synthesis were markedly inhibited only after 48 hours. Nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) levels decreased after 3 days. Cell membrane integrity was lost after 4 days. These results demonstrate that cells can be lethally damaged, yet continue to undergo apparently normal metabolic activities for several days. In a previous study, DNA double-strand breaks were detected after 1 day. We now show that by 2 days, breaks are visible as fragmentation in the nucleosome spacer regions of chromatin. This type of damage is consistent with cell death occurring by the process of apoptosis. Cell shrinkage and morphology were also consistent with this type of cell death. The slow cell death reported here appears to occur at the G2/M transition and may involve events that normally occur at this stage of the cell cycle. These results demonstrate the importance of DNA degradation as an early and possibly essential step in cell death.

Diet, Physical Activity, and Colorectal Cancer Among Chinese in North America and China

Abstract: In a population-based case-control study of colorectal cancer among Chinese men and women in western North America and the People's Republic of China, a common protocol was used to assess past life-style characteristics of 905 cases diagnosed during 1981-1986 and 2,488 controls. Risks for cancers of both the colon and rectum increased with increased food energy from fat, protein, carbohydrate, and all energy sources combined, for both sexes and on both continents. Yet, in multivariate analysis, colorectal cancer risk was significantly associated only with saturated fat; no relationships were seen with other dietary sources of energy. Colon cancer risk was elevated among men employed in sedentary occupations. On both continents and in both sexes, risks for cancers of both the colon and rectum increased with increasing time spent sitting. Further, the association between colorectal cancer risk and saturated fat was stronger among the sedentary than among the active. Risk among sedentary Chinese Americans of either sex increased more than fourfold from the lowest to the highest category of saturated fat intake. Among migrants to North America, risk increased with increasing years lived in North America. These observations suggest (a) that colorectal cancer risk increases with duration of exposure to a sedentary life-style and a diet rich in saturated fat; (b) that higher incidence among Chinese-American men relative to women is due to longer duration of these habits among men, who have lived longer in North America; and (c) that higher risk among Chinese Americans of both sexes relative to risk among the general population in China is due to differences in such habits. Attributable risk calculations suggest that, if these associations are causal, saturated fat intakes exceeding 10 g/day, particularly in combination with physical inactivity, could account for 60% of colorectal cancer incidence among Chinese-American men and 40% among Chinese-American women.

Prospective Study of the Frequency and Size Distribution of Polyps Missed by Colonoscopy

Abstract: An important determinant in interpreting the results of colorectal polyp chemoprevention trials is the rate of polyps missed during colonscopic examination. We prospectively examined 90 patients by tandem colonoscopy performed by two alternating examiners. In 69 (76.7%) patients, 221 neoplastic lesions were documented histologically. Of a total of 58 lesions detected in 31 patients, no neoplastic lesion greater than or equal to 10 mm in size was missed; 16% of diminutive (less than or equal to 5 mm) neoplastic polyps and 12.3% of medium-sized (6-9 mm) neoplastic polyps were missed by the first examiner. We conclude that an experienced colonoscopist will miss about 15% of colorectal neoplastic polyps less than 10 mm in size in the setting of adequate bowel preparation. Large (greater than or equal to 10 mm) polyps were rarely missed, however, with the "miss" rate in our study equal to 0, with a 95% confidence limit of 4.64%.

In Vivo Tumor Targeting of a Recombinant Single-Chain Antigen-Binding Protein

Abstract: We describe here the first in vivo targeting of tumors with a single-chain antigen-binding protein. The molecule, which was constructed and expressed in Escherichia coli, is a novel recombinant protein composed of a variable light-chain (VL), amino acid sequence of an immunoglobulin tethered to a variable heavy-chain (VH) sequence by a designed peptide. We show that this protein, derived from the DNA sequence of the variable regions of the antitumor monoclonal antibody B6.2, has the same in vitro antigen-binding properties as the B6.2 Fab' fragment. Comparative pharmacokinetic studies in athymic mice demonstrate much more rapid alpha and beta phases of plasma clearance for the single-chain antigen-binding protein than for the Fab' fragment, as well as an extremely rapid whole-body clearance. Half-life values for alpha and beta phases of single-chain antigen-binding protein clearance were 2.4 minutes and 2.8 hours, respectively, versus 14.8 minutes and 7.5 hours for Fab'. Furthermore, the single-chain antigen-binding protein molecule did not show accumulation in the kidney as did the Fab' molecule or, as previously shown, the F(ab')2 molecule. Despite its rapid clearance, the single-chain antigen-binding protein showed uptake in a human tumor xenograft comparable to that of the Fab' fragment, resulting in tumor to normal tissue ratios comparable to or greater than those obtained with the Fab' fragment. These studies thus demonstrate the in vivo stability of recombinant single-chain antigen-binding proteins and their potential in some diagnostic and therapeutic clinical applications in cancer and other diseases.

Expression of CYP1A1 Gene in Patients With Lung Cancer: Evidence for Cigarette Smoke-Induced Gene Expression in Normal Lung Tissue and for Altered Gene Regulation in Primary Pulmonary Carcinomas

Abstract: The major polycyclic aromatic hydrocarbon inducible-cytochrome P4501A1 gene (CYP1A1) is presumed to be important in pulmonary carcinogenesis and toxicology because its product, the cytochrome P4501A1-dependent (CYP1A1-dependent) monooxygenase, transforms selected xenobiotics (including polycyclic aromatic hydrocarbon procarcinogens in cigarette smoke) to potent carcinogenic metabolites. CYP1A1 messenger RNA (mRNA) expression has not, however, been previously demonstrated in human pulmonary tissue. This report defines CYP1A1 gene expression in normal lung tissue and primary pulmonary carcinoma tissue obtained at thoracotomy from 56 patients with lung cancer. When Northern blot hybridization analyses were performed, 17 of 19 (89%) and zero of five (0%) samples of normal lung tissue from active cigarette smokers and nonsmokers, respectively, expressed the normal 2.8-kilobase CYP1A1 mRNA. In addition, a time-dependent decrease in expression of the CYP1A1 gene was noted in normal lung tissue from individuals who were former smokers, with a decrease in expression occurring as early as 2 weeks following cessation of cigarette smoking. Expression became undetectable in all patients who had stopped smoking more than 6 weeks prior to study. When CYP1A1 gene expression was evaluated in lung cancers, mRNA levels were detectable in one of four (25%) tumors from nonsmokers; two of 24 (8%) tumors from former smokers; and seven of 15 (47%) tumors from cigarette smokers. In addition, an approximately 10-kilobase CYP1A1 RNA species, which was not detectable in normal lung tissue, was observed in five of ten (50%) of the lung cancers that expressed the CYP1A1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Controlled Trial of Megestrol Acetate for the Treatment of Cancer Anorexia and Cachexia

Abstract: Preliminary information has suggested that megestrol acetate leads to appetite stimulation and nonfluid weight gain in patients with breast cancer, other cancers, and AIDS. Pursuant to this, we developed a randomized, double-blind, placebo-controlled trial of megestrol acetate in patients with cancer-associated anorexia and cachexia. We randomly assigned 133 eligible patients to receive 800 mg of megestrol acetate per day or a placebo. Patients assigned to megestrol acetate more frequently reported improved appetite (P = .003) and food intake (P = .009) when compared with patients receiving the placebo. A weight gain of 15 lb or more over baseline was seen in 11 of 67 (16%) patients receiving megestrol acetate compared with one of 66 (2%) given the placebo (P = .003). Patients receiving megestrol acetate reported significantly less nausea (13% vs. 38%; P = .001) and emesis (8% vs. 25%, P = .009). No clinically or statistically significant toxic reactions were ascribed to megestrol acetate, with the exception of mild edema. This study convincingly demonstrated that megestrol acetate can stimulate appetite and food intake in patients with anorexia and cachexia associated with cancer, leading to significant weight gain in a proportion of such patients.

Effects of Tamoxifen Therapy on Lipid and Lipoprotein Levels in Postmenopausal Patients With Node-Negative Breast Cancer

Abstract: We conducted a 2-year, randomized, double-blind, placebo-controlled toxicity trial of therapy with tamoxifen (10 mg twice a day) in 140 postmenopausal women with a history of breast cancer and histologically negative axillary lymph nodes. These women had been treated with surgery with or without radiotherapy. At a 3-month evaluation, tamoxifen-treated women showed a significant decrease in fasting plasma levels of total cholesterol and low-density lipoprotein (LDL) cholesterol, which persisted at 6- and 12-month evaluations. During the first 12 months, plasma triglyceride levels increased; small but significant decreases in high-density lipoprotein cholesterol (HDL) were observed in tamoxifen-treated women, but ratios of total cholesterol to HDL cholesterol and of LDL to HDL cholesterol changed favorably. While data relating lipid/lipoprotein profiles and cardiovascular disease are limited in women, current evidence suggests that total cholesterol and possibly low-density lipoprotein cholesterol are risk factors. We conclude that during the first 12 months of treatment, tamoxifen exerts a favorable effect on the lipid profile in postmenopausal women with early stage breast cancer.

Effect of Tamoxifen on Serum Insulinlike Growth Factor I Levels in Stage I Breast Cancer Patients

Abstract: Insulinlike growth factor I (IGF-I) has been shown to be a potent mitogen for breast cancer cells in vitro, and IGF-I receptors have been demonstrated on human primary breast neoplasms. In a randomized, placebo-controlled study, we document that administration of the antiestrogen tamoxifen to patients with breast cancer was associated with a statistically significant (P = .002) reduction in the serum level of IGF-I. The mean IGF-I level was 1.4 U/mL in the placebo-treated group and 0.9 U/mL in the tamoxifen-treated group. Because serum IGF-I level is growth hormone (GH) dependent and because data suggest that the pubertal surge in GH and IGF-I levels is sex steroid dependent, we speculate that the mechanism underlying our observation may involve blockade by tamoxifen of estrogen action in the hypothalamic-pituitary axis. We conclude that tamoxifen treatment reduces IGF-I levels and that this reduction may contribute to the therapeutic effect of the drug.

Serum selenium and subsequent risk of cancer among Finnish men and women.

Abstract: The association between the serum selenium level and the subsequent incidence of cancer was investigated in a longitudinal study of 39,268 men and women participating in the Social Insurance Institution's Mobile Clinic Health Examination Survey in Finland The baseline examinations, including the collection of blood samples, were performed in 1968-1972 During a median follow-up of 10 years, 1,096 new cancer cases were identified from the files of the Finnish Cancer Registry Selenium concentrations were measured from the stored serum samples collected from these cancer cases and from two controls per case, matched for sex, municipality, and age The mean serum selenium level was 591 micrograms/L among all male cancer cases and 625 micrograms/L among controls The difference was statistically significant (P less than 001) Corresponding values among women were 636 and 639 micrograms/L, respectively Low serum selenium levels were associated with an increased risk of developing cancer at several sites, especially cancers of the stomach and lung among men The relative risk of lung cancer between the highest and lowest decile of serum selenium was 011, and it differed significantly from unity (P less than 001) These findings are in agreement with the hypothesis that low selenium intake may increase the risk of some cancers among men

Evidence for mendelian inheritance in the pathogenesis of lung cancer.

Abstract: Segregation analyses that allowed for variable age of onset of lung cancer and smoking history were performed on 337 families, each ascertained through a lung cancer proband. Results indicated compatibility of the data with mendelian codominant inheritance of a rare major autosomal gene that produces earlier age of onset of the cancer. Segregation at this putative locus could account for 69% and 47% of the cumulative incidence of lung cancer in individuals up to ages 50 and 60, respectively. The gene was involved in only 22% of all lung cancers in persons up to age 70, a reflection of an increasing proportion of noncarriers succumbing to the effects of long-term exposure to tobacco.

Increasing annual incidence of primary malignant brain tumors in the elderly.

Abstract: Between 1973 and 1985, total age-adjusted cancer incidence in the United States (all races, men and women) rose by 107%, with an average annual percentage change of +09% Analysis of reported age-specific incidence of primary malignant brain tumors over the same years demonstrates that incidence rates increased dramatically between 1973/1974 and 1985 In 1985, incidence rates for persons aged 75-79, 80-84, and 85 years of age and over were 187%, 394%, and 501%, respectively, of rates in 1973/1974 Similar increases were found in both men and women, analyzed separately and combined Average annual percentage changes in primary brain tumor incidence were +70%, +204%, and +234% in these age ranges, respectively Reported incidence in younger persons varied little over the same period of time The most common histologic type of primary brain tumor in the elderly was of glial origin, predominantly the glioblastoma multiforme and astrocytoma These tumors are highly malignant and invariably fatal Two possible causes may explain the increased incidence in the elderly: the introduction and extensive use of x-ray computed tomography since 1973 and/or a true increase in incidence occurring independently of diagnostic advances

Inhibition of Collagenolytic Activity and Metastasis of Tumor Cells by a Recombinant Human Tissue Inhibitor of Metalloproteinases

Abstract: Metalloproteinases secreted by tumor cells play an important role in metastasis. In the present study, we determined whether an inhibitor of these proteinases could inhibit the ability of tumor cells to degrade collagen and to metastasize. Metalloproteinases with degradative activities for type I collagen, type IV collagen, gelatin, and casein were secreted by a highly metastatic rat embryo cell line (4R) transfected by c-Ha-ras1 (also known as HRAS1). These metalloproteinases were identified by sodium dodecyl sulfate substrate-polyacrylamide gel electrophoresis as 92-kilodalton and 68-kilodalton gelatinolytic enzymes and 48-kilodalton and 45-kilodalton caseinolytic proteinases. A recombinant human tissue inhibitor of metalloproteinases (rTIMP) completely inhibited the proteolytic activities of these enzymes and was also a potent inhibitor of the proteolytic degradation of collagen by intact c-Ha-ras1-transfected cells. The ability of these cells to colonize the lungs after intravenous injection into nude mice was inhibited by 83% when rTIMP was repeatedly injected intraperitoneally into the animals. These data demonstrate that rTIMP is a potent inhibitor of the metalloproteinase activities of these cells and can also inhibit their metastatic potential.

Influence of Organ Environment on Extracellular Matrix Degradative Activity and Metastasis of Human Colon Carcinoma Cells

Abstract: Orthotopic implantation of human colon carcinoma cells is useful for studying the behavior of metastatic subpopulations. We observed that the parental line and variants of human colon carcinoma KM12 cells were all tumorigenic following implantation into the subcutis or cecal wall of BALB/c nude mice. Their ability to metastasize to distant organ sites varied, however, with the site of growth. Subcutaneous (SC) tumors did not produce visceral metastases, whereas cecal tumors metastasized to the regional mesenteric lymph nodes and to the liver. To examine the influence of organ environment on the extracellular matrix-degrading activity of the tumors, we inoculated human colon carcinoma cells into the subcutis or cecal wall and after 7 weeks isolated and cultured the tumors in serum-free medium. The conditioned media of SC tumors contained very low levels of type IV collagenase (gelatinase) and heparanase (heparan sulfate-specific endo-beta-D-glucuronidase), whereas the media of the cecal wall tumors contained high levels of both. Zymograms of the media revealed that the intracecal human colon carcinomas secreted more than three times the amount of latent and active forms of 92-kd type IV collagenase than did the SC tumors. Moreover, only the conditioned media of intracecal tumors contained latent and active forms of 64-kd type IV collagenase. Histochemical analysis using rabbit antiserum raised against the synthetic peptides of 72-kd procollagenase type IV showed type IV collagenase in the intracecal tumors; human colon carcinoma growing SC, however, were not stained significantly. These results suggest that factors in the organ environment may affect production and secretion of tumor extracellular matrix-degrading enzymes, and these factors may modify the metastatic behavior of human colon carcinoma cells in nude mice.

Phase I trial of 5-day continuous venous infusion of oxaliplatin at circadian rhythm-modulated rate compared with constant rate.

Abstract: The toxic effects and tissue uptake of both cisplatin and oxaliplatin--[(1R, 2R)-1,2-cyclohexanediamine-N,N'] [oxalato(2-)-O,O']platinum--were previously shown to vary similarly according to dosing time in mice. A 4-hour infusion of cisplatin resulted in fewer side effects and allowed administration of higher doses at 16 hours than at 4 hours in patients with cancer. We hypothesized that the continuous venous infusion of oxaliplatin for 5 days would be less toxic and would deliver a higher dose to the patient if the drug were infused at a circadian rhythm-modulated rate (peak at 16 hr; schedule B) rather than at a constant rate (schedule A). We tested this hypothesis in a randomized phase I trial. We escalated the dose of oxaliplatin to the patient by 25 mg/m2 per course. Courses were repeated every 3 weeks. An external, multichannel, programmable-in-time pump was used for the infusions. Toxicity was assessable for 94 courses in 23 patients (12 patients with breast carcinoma, nine with hepatocellular carcinoma, and two with cholangiocarcinoma). The incidence of neutropenia of World Health Organization grades II-IV and the incidence of distal paresthesias were 10 or more times higher (P less than .05) with schedule A than with schedule B. In addition, vomiting was 55% higher (P = .15) with schedule A than with schedule B. Furthermore, with schedule B, the mean dose of oxaliplatin (P less than .001) and its maximum tolerated dose (P = .06) could be increased by 15% over those doses with schedule A. An objective response was achieved in two of the 12 patients with previously treated breast cancer. We recommend that the dose of oxaliplatin for phase II trials be 175 mg/m2, delivered according to the circadian rhythm-modulated rate.

Serologic Precursors of Cancer. Retinol, Carotenoids, and Tocopherol and Risk of Prostate Cancer

Abstract: We investigated the associations of serum retinol, the carotenoids beta-carotene and lycopene, and tocopherol (vitamin E) with the risk of prostate cancer in a nested case-control study. For the study, serum obtained in 1974 from 25,802 persons in Washington County, MD, was used. Serum levels of the nutrients in 103 men who developed prostate cancer during the subsequent 13 years were compared with levels in 103 control subjects matched for age and race. Although no significant associations were observed with beta-carotene, lycopene, or tocopherol, the data suggested an inverse relationship between serum retinol and risk of prostate cancer. We analyzed data on the distribution of serum retinol by quartiles, using the lowest quartile as the reference value. Odds ratios were 0.67, 0.39, and 0.40 for the second, third, and highest quartiles, respectively.

Expression of mdr1 and mdr3 Multidrug-resistance Genes in Human Acute and Chronic Leukemias and Association With Stimulation of Drug Accumulation by Cyclosporine

Abstract: We determined the expression levels of the mdr1 and mdr3 multidrug-resistance genes (also known as PGY1 and PGY3, respectively) in peripheral blood cells from 69 adult patients with acute and chronic leukemias, using an RNase protection assay. Expression of mdr1 was found in samples from patients with acute nonlymphocytic leukemia (13 of 17), chronic myelocytic leukemia (CML, chronic phase, 10 of 10; blast crisis, three of four), acute lymphocytic leukemia (ALL, eight of 11), B-cell chronic lymphocytic leukemia (B-CLL, 17 of 17), hairy cell leukemia (HCL, one of two), and T-cell prolymphocytic leukemia (one of one), but not in B-cell prolymphocytic leukemia (B-PLL, 0 of seven). Expression of mdr3 was only detected in samples from B-cell lymphocytic leukemias: CML, lymphoid blast crisis (one of one), B-cell ALL (two of two), B-CLL (17 of 17), B-PLL (seven of seven), and HCL (two of two). In vitro drug uptake studies by on-line flow cytometry showed that in leukemia cells expressing either mdr1 or mdr3, the steady-state accumulation of daunorubicin could be significantly increased by addition of cyclosporine and, to a lesser extent, by verapamil. Because cyclosporine and verapamil are known as inhibitors of the mdr1-encoded P-glycoprotein drug-efflux pump, and because the mdr1 and mdr3 genes are highly homologous, our data suggest that the mdr3 gene encodes a functional drug pump in B-cell lymphocytic leukemias. The results of this study may have implications for clinical therapy for acute or chronic leukemias expressing the mdr1 or mdr3 gene, in particular, treatment with combinations of cytotoxic drugs plus agents that reverse multidrug resistance. Since mdr1 and mdr3 are frequently expressed in untreated as well as treated leukemia, such combination therapy should be considered for untreated patients as well as treated patients.

Dictyostelium nucleoside diphosphate kinase highly homologous to Nm23 and Awd proteins involved in mammalian tumor metastasis and Drosophila development.

Abstract: Two complementary DNAs (cDNAs) previously isolated, one by functional screening and the other by immunological screening of a Dictyostelium discoideum expression library, encode two proteins, Gip17 and Guk7.2, sharing 71% homology. In the present study, we found that the expression of their messenger RNAs (mRNAs) is developmentally regulated, with a sharp decrease during the first hours of differentiation. The Gip17 protein was purified to homogeneity from D. discoideum amoebas and from recombinant Escherichia coli and was conclusively identified as a nucleoside diphosphate (NDP) kinase. NDP kinases play a major role in synthesis of nucleoside triphosphates and, in many systems, are found associated with guanosine triphosphate (GTP)-binding proteins. We found the Gip17 protein to be 77% homologous to the human Nm23 protein and 75% homologous to the Drosophila melanogaster Awd protein. The levels of murine and human nm23 mRNA and Nm23 protein are significantly reduced in tumor cells of high metastatic potential, suggesting that Nm23 is involved in suppression of mammalian tumor metastasis, and mutants of the awd gene exhibit widespread development abnormalities, suggesting that Awd is involved in D. melanogaster development. The high percentage of homology of the Gip17 and Guk7.2 proteins with the Nm23 and Awd proteins indicates that Nm23 and Awd also have nucleoside diphosphate kinase activity. Possible modulations in the activity of this metabolic enzyme could be related to the altered metabolism of tumor cells and the control of metastatic potential. Our results point to an unexpected role of NDP kinase in development, growth control, and oncogenic transformation.