Showing papers in "Cancer Research in 1990"

Isolation and Characterization of a Spontaneously Immortalized Human Breast Epithelial Cell Line, MCF-10

Abstract: Two sublines of a breast epithelial cell culture, MCF-10, derived from human fibrocystic mammary tissue exhibit immortality after extended cultivation in low calcium concentrations (0.03-0.06 mM) and floating transfers in low calcium (MCF-10F), or by trypsin-Versene passages in the customary (normal) calcium levels, 1.05 mM (MCF-10A). Both sublines have been maintained as separate entities after 2.3 years (849 days) in vitro and at present have been in culture for longer than 4 years. MCF-10 has the characteristics of normal breast epithelium by the following criteria: (a) lack of tumorigenicity in nude mice; (b) three-dimensional growth in collagen; (c) growth in culture that is controlled by hormones and growth factors; (d) lack of anchorage-independent growth; and (e) dome formation in confluent cultures. Cytogenetic analysis prior to immortalization showed normal diploid cells; although later passages showed minimal rearrangement and near-diploidy, the immortal cells were not karyotypically normal. The emergence of an immortal culture in normal calcium media was not an inherent characteristic of the original tissue from which MCF-10 was derived since reactivated cryo-preserved cells from cultures grown for 0.3 and 1.2 years in low calcium were incapable of sustained growth in normal calcium.

Critical Factors in the Biology of Human Cancer Metastasis: Twenty-eighth G. H. A. Clowes Memorial Award Lecture

Abstract: The process of metastasis is not random. Rather, it consists of a series of linked, sequential steps that must be completed by tumor cells if a metastasis is to develop. Although some of the steps in this process contain stochastic elements, as a whole, metastasis favors the survival and growth of a few subpopulations of cells that preexist within the parent neoplasm. Moreover, metastases can have a clonal origin, and different metastases can originate from the proliferation of single cells. The outcome of metastasis depends on the interaction of metastatic cells with different organ environments. Organ-specific metastases have been demonstrated in a variety of experimental tumor systems. Moreover, we have found tumor growth that is specific to a particular site within one organ. Whether the same conclusions can be reached for human cancers remained unanswered until very recently. Studies from our laboratory and from others have shown that the implantation of human cancer cells derived from surgical specimens into correct anatomical sites of nude mice can provide a suitable model of metastasis of human tumors. Clonal analysis of a human renal carcinoma, colon carcinomas, and melanomas has revealed that these tumors are indeed heterogeneous for metastatic properties, an observation made only after orthotopic implantation. Thus, growth in the environment of specific organs can be selective and the environment per se influences this process. While it is clear that vascularity and local immunity can facilitate or retard tumor growth, we have concentrated on understanding how damage to an organ and the subsequent repair process can facilitate tumor cell proliferation. Accelerated growth of human colon cancer cells was found in hepatectomized nude mice, whereas accelerated growth of human renal cancer cells was found in nephrectomized nude mice. These data suggest that systemic physiological signals can be recognized by neoplastic cells presumably by mechanisms similar to those shared by their normal cell counterparts. In summary, the critical factors that regulate metastasis are the intrinsic properties of metastatic cells and host factors involved in homeostasis. The recent increase in our understanding of metastasis should provide important leads for developing more effective approaches to the treatment of disseminated cancer.

p53 gene mutations occur in combination with 17p allelic deletions as late events in colorectal tumorigenesis.

Abstract: Coordinate loss of one copy of the p53 gene and mutation of the remaining copy occur in colorectal carcinomas and in many other human malignancies. However, the prevalence of p53 gene mutations in carcinomas which maintain both parental copies of p53 has not previously been evaluated. Moreover, it is not known whether p53 gene mutations are limited to malignant tumors or whether they can also occur in benign neoplasms. To answer these questions, a total of 58 colorectal tumors have been examined; in each tumor, allelic losses were assessed using restriction fragment length polymorphisms and p53 gene mutations were assessed by sequencing cloned polymerase chain reaction products. The following conclusions emerged: (a) p53 gene mutations occurred but were relatively rare in adenomas, regardless of size and whether the adenomas were derived from patients with familial adenomatous polyposis; (b) In carcinomas as well as in adenomas, p53 gene mutations were infrequently observed in tumors which contain both copies of chromosome 17p (17% of 30 tumors), while tumors which lost one copy of chromosome 17p usually had a mutation in the remaining p53 allele (86% of 28 tumors); (c) p53 gene mutations were found at similar frequencies in primary tumor samples and in cell lines derived from tumors. These and other data suggest that the rate limiting step in p53 inactivation is point mutation and that once a mutation occurs, loss of the remaining wild-type allele rapidly follows. Both mutations and allelic losses generally occur near the transition from benign to malignant growth, and the p53 gene may play a causal role in this progression.

Increased cell division as a cause of human cancer

Abstract: Carcinogenesis research is increasingly focused on chemicals that are not genotoxic and yet, at high doses, can induce cancer, apparently by increasing cell proliferation. We hypothesize that increased cell division per se stimulated by external or internal factors is also associated with the development of many human cancers. Although this hypothesis is well substantiated in the experimental literature, it has not been generalized as an important mechanism for carcinogenesis in human populations. Under this increased cell division model, the pathogenesis of cancer may result from molecular genetic errors induced during the process of cell division and from altered growth control of malignant or premalignant cells. Molecular genetic analysis of human cancers has shown that tumor cells contain multiple genetic defects including mutations, translocations, and amplifications of oncogenes and are reduced to homozygosity for putative tumor suppressor genes; these phenomena all require cell division for their occurrence and fixation. Increased cell division increases the risk of such events occurring. An accumulation of a combination of such genetic errors leads to a neoplastic phenotype. Examples are discussed of human cancers in which increased cell division, which drives the accumulation of genetic errors and can lead to neoplastic transformation, is caused by hormones, drugs, infectious agents, chemicals, physical or mechanical trauma, and other chronic irritation.

Integrin distribution in malignant melanoma: association of the beta 3 subunit with tumor progression

Abstract: Since tumor progression is dependent on the ability of malignant cells to interact with the extracellular matrix, molecules on the cell surface which mediate cell-substratum interactions are likely to be important regulators of tumor invasion and metastasis The purpose of this study was to examine the distribution of one such group of cell adhesion receptors, the integrins, in benign and malignant lesions of human melanocytes The distribution of integrin adhesion receptors was defined on cells in culture derived from normal and malignant melanocytes and in tissue sections from benign to increasingly malignant melanocytic lesions using a panel of monoclonal antibodies against specific integrin subunits Cells in culture expressed a large variety of integrins, including all of the previously characterized members of the beta 1 subfamily plus the alpha v/beta 3 vitronectin receptor The expression of integrins was similar in cells cultured from either benign or malignant lesions In contrast, consistent differences were noted in integrin expression by cells within tissues containing metastatic and vertical growth phase melanomas when compared to radial growth phase melanoma cells and cells within nevi Most notably, the expression of the beta 3 subunit was restricted exclusively to cells within vertical growth phase and metastatic melanomas The presence of this integrin may be important in the development of tumor invasiveness and could be useful as a marker of melanoma cells entering the more aggressive phase of the malignant process

Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer.

Abstract: Previous studies have suggested that overexpression of HER-2/neu oncogene occurs in 15-40% of breast cancers and that overexpression is associated with poor prognosis. In the present report, we have used an immunohistochemical technique involving a monoclonal antibody specifically reactive with the external domain of HER-2/neu to study expression of HER-2/neu in frozen sections of normal ovary and advanced epithelial ovarian cancer. The intensity of staining for HER-2neu was always moderate or less (0-2+) in normal ovarian epithelium. Among 73 ovarian cancers, 50 (68%) had staining similar to that for normal ovarian epithelium (0-2+) while 23 (32%) stained heavily (3+). Survival of the 23 patients with high HER-2/neu expression (median, 15.7 months) was significantly worse (P = 0.001) than that of the 50 patients (median, 32.8 months) with normal HER-2/neu expression. In addition, patients whose tumors had high HER-2/neu expression were significantly less likely to have a complete response to primary therapy (P less than 0.05) or have a negative second-look laparotomy when serum CA 125 levels were normal preoperatively (P less than 0.05). These findings suggest that HER-2/neu deserves further evaluation as a prognostic marker in epithelial ovarian cancer.

Interstitial Pressure Gradients in Tissue-isolated and Subcutaneous Tumors: Implications for Therapy

Abstract: High interstitial fluid pressure (IFP) in solid tumors is associated with reduced blood flow as well as inadequate delivery of therapeutic agents such as monoclonal antibodies. In the present study, IFP was measured as a function of radial position within two rat tissue-isolated tumors (mammary adenocarcinoma R3230AC, 0.4-1.9 g, n = 9, and Walker 256 carcinoma, 0.5-5.0 g, n = 6) and a s.c. tumor (mammary adenocarcinoma R3230AC, 0.6-20.0 g, n = 7). Micropipettes (tip diameters 2 to 4 microns) connected to a servo-null pressure-monitoring system were introduced to depths of 2.5 to 3.5 mm from the tumor surface and IFP was measured while the micropipettes were retrieved to the surface. The majority (86%) of the pressure profiles demonstrated a large gradient in the periphery leading to a plateau of almost uniform pressure in the deeper layers of the tumors. Within isolated tumors, pressures reached plateau values at a distance of 0.2 to 1.1 mm from the surface. In s.c. tumors the sharp increase began in skin and levelled off at the skin-tumor interface. These results demonstrate for the first time that the IFP is elevated throughout the tumor and drops precipitously to normal values in the tumor's periphery or in the immediately surrounding tissue. These results confirm the predictions of our recently published mathematical model of interstitial fluid transport in tumors (Jain and Baxter, Cancer Res., 48: 7022-7032, 1988), offer novel insight into the etiology of interstitial hypertension, and suggest possible strategies for improved delivery of therapeutic agents.

Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents.

Abstract: Alkylating agents and compounds converted metabolically to alkylating agents form an important class of carcinogens and mutagens that includes a number of environmentally important /V-nitrosocompounds. A number of alkylating agents are also useful antitumor drugs. There are many factors determining the sensitivity to the toxic, mutagenic, and carcinogenic poten tial of these compounds (1-4).2 One of these is the capacity to

Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid.

Abstract: Grilled ground beef contains factors that inhibit the initiation of mouse epidermal carcinogenesis by 7,12-dimethylbenz(a)anthracene. Previously we isolated an active principal and characterized it as an isomeric mixture of conjugated dienoic derivatives of linoleic acid (CLA). We now show that synthetic CLA inhibits the initiation of mouse forestomach tumorigenesis by benzo(a)pyrene. Four and 2 days prior to p.o. treatment with benzo(a)pyrene, female ICR mice were given (a) CLA in olive oil, (b) linoleic acid in olive oil, or (c) olive oil alone or plus 0.85% saline (control groups). Three days later the cycle was repeated for a total of 4 times. At 30 wk of age, the mice were sacrificed. In three independent experiments, mice treated with CLA developed only about half as many neoplasms/animal as mice in the control groups (P

Evaluation of the Antitumor Activity of Gemcitabine (2′,2′-Difluoro-2′-deoxycytidine)

Abstract: A new pyrimidine antimetabolite, 2',2'-difluorodeoxycytidine, Gemcitabine (LY188011, dFdCyd) has been synthesized and evaluated in experimental tumor models. dFdCyd is a very potent and specific deoxycytidine analogue. The concentration required for 50% inhibition of growth is 1 ng/ml in the CCRF-CEM human leukemia cell culture assay. Concurrent addition of deoxycytidine to the cell culture system provides about a 1000-fold decrease in biological activity. The inhibition of growth of human leukemia cells in culture led to the in vivo evaluation of this compound as a potential oncolytic agent. Maximal activity in vivo was seen with dFdCyd when administered on an every third day schedule. 1-beta-D-Arabinofuranosylcytosine, administered on a daily for 10-day schedule, was directly compared to dFdCyd in this evaluation. dFdCyd demonstrated good to excellent antitumor activity in eight of the eight murine tumor models evaluated. 1-beta-D-Arabinofuranosylcytosine was substantially less active or had no activity in these same tumor models. This in vivo activity against murine solid tumors supports the conclusion that dFdCyd is an excellent candidate for clinical trials in the treatment of cancer.

Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice.

Abstract: There are few reports describing experimental models of the growth and metastasis of human breast carcinomas. This article discusses the tumorigenic and metastatic properties of two estrogen receptor-negative breast carcinomas injected into nude mice. Tumor growth in the mammary fatpad (m.f.p.) and the subcutis was compared in female nude mice. The injection of 10(5) viable cells of two human breast carcinoma cell lines (MDA-MB-231 and MDA-MB-435) gave a 100% tumor take rate in the m.f.p., whereas only 40% of the s.c. injections produced tumors and these occurred several weeks after the appearance of the m.f.p. tumors. Thus, the m.f.p. of nude mice is a favorable site for the growth of human breast carcinomas. MDA-MB-435 tumors produced distant metastases in 80% to 100% of recipients. The most common sites for metastasis were the lymph nodes and lungs, with a lower incidence of metastases in muscle (chest wall and thigh), heart, and brain. New variant cell lines were isolated from metastases in the lungs, brain, and heart. All the cell lines were tumorigenic in the m.f.p., and the lung- and heart-derived metastasis lines produced slightly more lung metastases than the original cell line. However, the brain metastasis variant produced significantly fewer lung metastases. Intravenous inoculation of the spontaneous metastasis-derived cell lines produced few lung colonies. Only cell variants isolated from experimental lung metastases showed enhanced lung colonization potential when reinjected i.v. Our results suggest that the estrogen receptor-negative MDA-MB-435 cell line injected in the m.f.p. of nude mice could be a valuable tool for analysis of the cellular and molecular basis of the metastasis of advanced breast cancer.

Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors.

Abstract: The efficacy in cancer treatment of monoclonal antibodies or other macromolecules bound to radionuclides, chemotherapeutic agents, toxins, enzymes, growth factors, or effector antibodies has been limited by their inability to reach their target in vivo in adequate quantities. Heterogeneity of tumor-associated antigen expression alone has failed to explain the nonuniform uptake of antibodies. As a result, only in recent years have the peculiarities of tumor physiology been recognized as determinants of antibody distribution. Three physiological barriers responsible for the poor localization of macromolecules in tumors have been identified: (a) heterogeneous blood supply; (b) elevated interstitial pressure; and (c) large transport distances in the interstitium. The first barrier limits the delivery of blood-borne molecules to well-perfused regions of a tumor; the second barrier reduces extravasation of fluid and macromolecules in the high interstitial pressure regions and also leads to an experimentally verifiable, radially outward convection in the tumor periphery which opposes the inward diffusion; and the third barrier increases the time required for slowly moving macromolecules to reach distal regions of a tumor. Binding of antibody to an antigen further lowers the effective diffusion rate of the antibody by reducing the amount of mobile antibody. Due to micro- and macroscopic heterogeneities in tumors, the relative magnitude of each of these barriers would vary from one location to another and from one day to the next in the same tumor and from one tumor to another. If the genetically engineered macromolecules, e.g., lymphokines, and other new modalities, e.g., killer lymphocytes, as well as low molecular weight cytotoxic agents, are to fulfill their clinical promise, methods must be developed to overcome these physiological barriers. Some of these methods are discussed, and situations wherein these barriers may not be a problem are pointed out.

Smoking and drinking in relation to cancers of the oral cavity, pharynx, larynx, and esophagus in northern Italy

Abstract: A hospital-based case-control study of upper aerodigestive tract tumors was conducted between June 1986 and June 1989 in Northern Italy. One hundred fifty-seven male cases of oral cavity cancer, 134 of pharyngeal cancer, 162 of laryngeal cancer, and 288 of esophageal cancer, and 1272 male inpatients with acute conditions unrelated to tobacco and alcohol were interviewed. Odds ratios for current smokers of cigarettes were 11.1 for oral cavity, 12.9 for pharynx, 4.6 for larynx, and 3.8 for esophagus. For all 4 sites, the risk increased with increasing number of cigarettes and duration of smoking habits and, with the exception of esophageal cancer, decreased with increasing age at the start of and years since quitting smoking. Smokers of pipes and cigars showed a more elevated risk of cancer of the oral cavity and esophagus than did cigarette smokers. Significantly increased risks emerged also in heavy drinkers (odds ratio greater than 60 versus greater than or equal to 19 drinks/week = 3.4, 3.6, 2.1, and 6.0 for oral cavity, pharynx, larynx, and esophagus, respectively), deriving predominantly from wine consumption.

Characterization of Murine Monoclonal Antibodies Reactive to Either the Human Epidermal Growth Factor Receptor or HER2/neu Gene Product

Abstract: High levels of expression of either the epidermal growth factor receptor or the receptor-like HER2/neu gene product p185HER2 have been observed in a variety of human malignancies. Because of the association of this high level expression with certain human tumors, we have generated a panel of monoclonal antibodies specific for either the epidermal growth factor receptor or p185HER2 to study their structure, function, and antigenic domains in the normal and neoplastic states. We used the epidermoid carcinoma line A431 to generate five monoclonal antibodies which immunoprecipitate the epidermal growth factor receptor. These monoclonal antibodies bind to the extracellular domain of the epidermal growth factor receptor and demonstrate variable effects on epidermal growth factor binding. We used a stably transfected NIH 3T3 cell line expressing the HER2/neu gene to produce and characterize 10 monoclonal antibodies which immunoprecipitate p185HER2. These monoclonal antibodies bind to the extracellular domain of p185HER2 and do not cross-react with the epidermal growth factor receptor. The characteristics and potential applications of these monoclonal antibodies will be discussed.

Allelotype of Breast Cancer: Cumulative Allele Losses Promote Tumor Progression in Primary Breast Cancer

Abstract: Allele loss on a specific chromosome has implied the existence of a tumor suppressor gene such as the p53 gene and the RB gene. In order to determine which chromsome(s) carries a tumor suppressor gene(s) that contributes to tumor progression in primary breast cancer, we analyzed the loss of heterozygosity for each autosomal chromosome arm by using 39 restriction fragment length polymorphism markers including 25 variable numbers of tandem repeat probes. In 79 primary breast cancers, we found the frequent loss on the long arm of chromosome 13 (21%), the long arm of chromosome 16 (45%), and the short arm of chromosome 17 (56%). Interestingly, breast cancers in which loss of both chromosomes 13q and 17p was detected showed more malignant histopathological features, and a group of the tumors in which chromosome 16q loss was detected presented with frequent lymph node metastasis. Furthermore, the result of the deletion mapping on chromosome 17p implied the existence of a tumor suppressor gene distal to the p53 gene as well as the p53 gene itself for primary breast cancer. These results suggest that at least 4 tumor suppressor genes exist on chromosomes 13q, 16q, and 17p for primary breast cancer.

Purification and NH2-Terminal Amino Acid Sequence of Guinea Pig Tumor-secreted Vascular Permeability Factor

Abstract: Rodent and human tumor cell lines secrete a potent vascular permeability factor (VPF) which causes a rapid and substantial increase in microvascular permeability to plasma proteins without causing mast cell degranulation, or endothelial cell damage or without exciting an inflammatory cell infiltrate [D. R. Senger, S. J. Galli, A. M. Dvorak, C. A. Perruzzi, V. S. Harvey, and H. F. Dvorak. Science (Wash. DC), 219: 983-985, 1983; D. R. Senger, C. A. Perruzzi, J. Feder, and H.F. Dvorak. Cancer Res., 46: 5629-5632, 1986]. VPF now has been purified to homogeneity from guinea pig tumor cell culture medium; it is a Mr 34,000-43,000 protein, and a NH2-terminal amino acid sequence has been derived. A synthetic peptide corresponding to amino acid residues 1-24 of the native protein was used to raise rabbit antibodies which bind all of the vessel permeability-increasing activity secreted by guinea pig tumor cells and which stain purified VPF on immunoblots. These findings establish that this NH2-terminal amino acid sequence was derived from the permeability factor. Homology searches found no identity or close similarity between VPF NH2-terminal sequence and database sequences, indicating that VPF is distinct from other proteins for which sequence data are available. In particular, no sequence similarity was found between tumor-secreted VPF and other mediators of increased vessel permeability including plasma and glandular kallikreins.

Programmed cell death during regression of PC-82 human prostate cancer following androgen ablation.

Abstract: To study the mechanism of regression of human prostatic cancer following androgen ablation, the androgen-responsive PC-82 human prostatic adenocarcinoma xenograft was used as a model system Castration of male nude mice bearing PC-82 xenografts results in a 50% tumor regression by 2 wk following androgen ablation This regression is due to a sequence of biochemical and morphological events that results in both the cessation of cell proliferation and activation of programmed death or apoptosis of the androgen-dependent prostatic cancer cells Associated with this response are an enhanced expression of the transforming growth factor beta 1 gene, a potent inhibitor of cell proliferation, and testosterone-repressed prostatic message 2 (designated TRPM-2), a programmed cell death-associated gene Fragmentation of tumor DNA into nucleosomal oligomers and histological appearance of apoptotic bodies are characteristic early events that preceded the dramatic reduction in tumor volume following androgen ablation These results suggest that androgen-dependent human prostatic cancer cells, like normal prostatic cells, retain the ability to inhibit proliferation and to activate programmed cell death in response to androgen ablation Clarification of the biochemical pathway involved in the activation of this programmed cell death should identify new targets of therapy for even androgen-independent human prostatic cancer

p185neu Expression in Human Lung Adenocarcinomas Predicts Shortened Survival

Abstract: p185neu is the protein product of the HER2/neu protooncogene. This protein has characteristics of a tyrosine kinase growth factor receptor and is postulated to be important in human carcinogenesis. To define the significance of the expression of this protein in human non-small cell lung cancer, 55 tumors from patients with squamous cell carcinoma (16), adenocarcinoma (29), or large cell carcinoma (10) of the lung were examined for p185neu using immunohistological methods. Five of 16 squamous cell carcinomas and 10 of 29 adenocarcinomas were found to overexpress p185neu relative to levels of expression seen in uninvolved bronchiolar epithelium. For the adenocarcinomas, p185neu expression was associated with older age (66.6 +/- 10.1 versus 57.5 +/- 10.8 years) (P = 0.04) and shortened survival (83.7 +/- 94.1 versus 188.5 +/- 120 weeks) (P = 0.01). In this group, using Cox's multivariate survival analysis, p185neu expression was found to be a significant determinant of survival (P = 0.04) even after accounting for the effect of tumor stage. For the squamous cell carcinomas, p185neu expression was not correlated with any of our clinicopathological parameters. Our findings indicate that non-small cell lung cancers which express p185neu do so at levels higher than that found in normal bronchiolar epithelium, and expression in adenocarcinomas of the lung is independently associated with diminished survival intervals.

Retroviral vector-mediated γ-interferon gene transfer into tumor cells generates potent and long lasting antitumor immunity

Abstract: Retroviral vectors were used to introduce the γ-interferon (IFN-γ) gene into CMS-5 cells, a weakly immunogenic tumor of BALB/c origin. After selection in G418-containing medium, colonies were isolated, cloned, and expanded to cell lines. IFN-γ secretion was assessed using a bioassay and enzyme-linked immunosorbent assay, and high (25 units/ml) and low (5 units/ml) IFN-γ producers were isolated. Tumor growth was followed after intradermal injection, and spleen cells were isolated at different time points. IFN-γ secretion by tumor cells abrogated their tumorigenicity and induced a persistent and specific antitumor immunity. In contrast to the normally observed cellular immunosuppression in unmodified CMS-5 tumor-bearing mice, IFN-γ-producing tumors induced a long lasting state of T-cell immunity, as judged by rejection of a CMS-5 tumor challenge and persistence of specific cytotoxic activity in the spleen cell population. High levels of tumor-specific cytotoxic activity could also be detected if IFN-γ-secreting tumor cells, but not unmodified CMS-5 cells, were used as targets at a time point when immunosuppression was usually seen. These studies highlight the potential advantages of localized IFN-γ secretion to induce potent antitumor immune responses.

Relationship between dihydropyrimidine dehydrogenase activity and plasma 5-fluorouracil levels with evidence for circadian variation of enzyme activity and plasma drug levels in cancer patients receiving 5-fluorouracil by protracted continuous infusion

Abstract: The activity of dihydropyrimidine dehydrogenase (DPD) in peripheral blood mononuclear cells and plasma concentration of 5-fluorouracil (FUra) were simultaneously determined in cancer patients receiving FUra by protracted continuous infusion (300 mg/m2/day). Blood samples were drawn every 3 h over 24-h period and the resulting DPD and FUra values analyzed for circadian periodicity. In the seven patients studied, a circadian rhythm of DPD activity was observed (P less than 0.00001, Cosinor analysis) with the peak of activity at 1 a.m. (0.197 +/- 0.007 nmol/min/mg) and the trough at a 1 p.m. (0.113 +/- 0.007 nmol/min/mg). In addition, a circadian rhythm was observed for the plasma concentrations of FUra obtained over a 24-h period (P less than 0.00001, Cosinor analysis) with peak values (27.4 +/- 1.3 ng/ml) occurring at 11 a.m. and trough values (5.6 +/- 1.3 ng/ml) occurring at 11 p.m. The ratio of the maximum concentration of FUra to the minimum concentration observed was almost 5-fold. This study demonstrates a circadian variation of DPD activity in human peripheral blood mononuclear cells and a circadian variation of FUra plasma levels in patients receiving FUra by protracted continuous infusion. An inverse relationship between the circadian patterns of DPD activity and FUra plasma levels was also noted, suggesting that an association may exist between DPD activity and FUra plasma concentration. Further evidence of an association between DPD activity in peripheral blood mononuclear cells and plasma FUra concentration was demonstrated by a linear relationship between the two parameters in all patients (r = -0.627) and within individual patients (-0.978 less than r less than -0.742). With the recent advent of programmable pumps, information on the circadian pattern of FUra and/or DPD may be useful in planning continuous infusion schedules in order that optimal plasma drug concentration may be maintained over a 24-h cycle, thereby enhancing the therapeutic efficacy of FUra administered by continuous infusion.

HER-2/neu amplification predicts poor survival in node-positive breast cancer.

Abstract: HER-2/neu protooncogene amplification and protein expression were analyzed with slot blot and Western blot techniques, respectively, in more than 300 invasive primary breast tumors of all stages. Amplification (2- greater than 30 copies) was found in 17% of these tumors and high expression was seen in 19%. There was a striking coincidence between gene amplification and high expression. Tumors associated with many involved axillary lymph nodes or with Stage IV disease were more often HER-2/neu amplified or overexpressed. Furthermore, gene alteration was strongly correlated with the absence of steroid receptors and with larger tumor size. High expression without gene amplification was seen in a minor subset of tumors of less aggressive character. Neither amplification nor overexpression was correlated with disease outcome for patients with negative axillary lymph nodes. For node-positive patients, however, HER-2/neu amplification was a significant predictor of early relapse and death (median follow-up = 45 months), and a similar trend, although not significant, existed for high gene expression. Multivariate analyses indicated that HER-2/neu alterations were not independent predictors of patient outcome.

Involvement of Nucleic Acid Synthesis in Cell Killing Mechanisms of Topoisomerase Poisons

Abstract: The primary cytotoxic mechanism of camptothecin has been proposed to involve an interaction between the replication machinery and the camptothecin-mediated topoisomerase I-DNA cleavable complex (Y. H. Hsiang, M.G. Lihou, and L.F. Liu, Cancer Res., 49:5077-5082, 1989). In the present study, we show that killing of V79 cells by the topoisomerase II poisons 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide may involve ongoing RNA synthesis in addition to ongoing DNA synthesis. V79 cells synchronized by mitotic shake-off were treated with topoisomerase poisons in the presence of inhibitors of nucleic acid synthesis. S-Phase V79 cells were more sensitive to the topoisomerase I poison camptothecin and the topoisomerase II poison m-AMSA than G1-phase cells. The greater sensitivity of S-phase cells to killing by m-AMSA and camptothecin was abolished during cotreatment, but not posttreatment, with aphidicolin, suggesting that ongoing DNA synthesis in involved in cell killing by both topoisomerase I and II poisons. Cotreatment with transcription inhibitors, such as 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole or cordycepin, partially protected cells from the cytotoxic effects of m-AMSA but had no effect on camptothecin-mediated cytotoxicity. These results suggest that ongoing RNA transcription may be involved in cell killing by topoisomerase II poisons but not topoisomerase I poisons. Cotreatment with camptothecin reduced m-AMSA-mediated cytotoxicity in G1-phase V79 cells, suggesting a possible antagonism between topoisomerase I and II poisons. This antagonistic effect between topoisomerase I and II poisons could be explained by the strong inhibitory effect of camptothecin on RNA transcription.

Development of Antiestrogens and Their Use in Breast Cancer: Eighth Cain Memorial Award Lecture

Abstract: This paper describes the laboratory discovery and clinical testing of the first nonsteroidal antiestrogen, MER-25 (ethamoxytriphetol). The compound blocks estrogen action in all species tested and has only slight but transient estrogenic effects. No other antisteroidal actions are noted. MER-25 is antiestrogenic in primates and was investigated in the clinics in a wide range of gynecological conditions, including breast and endometrial cancer. Unfortunately toxic side effects (hallucinations, etc.) precluded further investigation. A derivative of triphenylethylene, clomiphene, has some partial agonist (estrogen-like) actions in laboratory animals and following clinical evaluation is now an established agent for the induction of ovulation in subfertile women. Although clomiphene is active in advanced breast cancer, it was not developed further. In the late 1960s a related compound, tamoxifen, was evaluated to treat a number of estrogen-responsive disorders but was successfully introduced in the 1970s for the treatment of advanced breast cancer. Although there was only modest initial interest in the palliative use of tamoxifen, an enormous increase in basic and applied studies with antiestrogens resulted in a definition of the target site-specific and tumoristatic actions of tamoxifen. Close cooperation between laboratory and clinical evaluation has guided the subsequent development of tamoxifen which is now available to treat all stages of breast cancer. Long-term adjuvant tamoxifen therapy, a concept developed in the laboratory, is currently the treatment strategy of choice. The considerable success of tamoxifen has focused attention on new antiestrogens with different pharmacological properties for other potential clinical applications.

Identification of candidate cancer chemopreventive agents and their evaluation in animal models and human clinical trials: a review.

Abstract: A search of the literature using National Library of Medicine databases and individual cancer journal articles yielded over 500 compounds with published chemopreventive activity in animals. From these, an initial 16 agents or agent combinations have been evaluated in the following animal tumor models: mouse skin papillomas/carcinomas induced by 7,12-dimethylbenz( a )anthracene/12- O -tetradecanoylphorbol-13-acetate; rat breast adenocarcinoma induced by N -methyl- N -nitrosourea or 7,12-dimethylbenz( a )anthracene; hamster lung carcinoma induced by N -methyl- N -nitrosourea or diethylnitrosamine; mouse bladder papillary carcinoma induced by N -butyl- N -(4-hydroxybutyl)nitrosamine; and rat and mouse colon cancer induced by azoxymethane/methylazoxymethanol acetate. Some of the most interesting positive results observed include 4-hydroxyphenyl retinamide plus tamoxifen in breast cancer, piroxicam in colon cancer, dimethylfluoroornithine in breast and bladder cancer, oltipraz in lung cancer, dehydroepiandrosterone in colon cancer, and molybdate in bladder cancer. Eighteen human intervention trials in progress are described that involve the following agents: β-carotene (eight trials). Retinol/retinoic acid (seven trials), vitamins C and E (three trials), 4-hydroxyphenyl retinamide (one trial), piroxicam (one trial), and calcium (one trial). By organ site these studies involve cancer of the lung (six studies), skin (five studies), colon (four studies), breast (one study), and uterine cervix (two studies).

Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis

Abstract: We established a continuous cell line, NCI-H295, from an invasive primary adrenocortical carcinoma. The cell line was established in a fully defined medium (HITES) and later could be adapted for growth in a simple medium supplemented only with selenium, insulin, and transferrin and devoid of serum, steroids, fibroblast growth factor, and a source of exogenous cholesterol. NCI-H295 cells had a relatively long population doubling time and were tumorigenic when inoculated s.c. into athymic nude mice. The cultured cells had ultrastructural features of steroid-secreting cells and contained complex cytogenetic abnormalities including the presence of multiple marker chromosomes. Steroid analyses (radioimmunoassays and mass spectrometry), performed 7 to 9 years after culture initiation, demonstrated secretion of more than 30 steroids characteristic of adrenocortical cells. Total unconjugated steroid secretion in serum-supplemented medium was 2.83 micrograms/10(6) cells/24 h and about 4-fold less in serum-free medium. The major pathway of pregnenolone metabolism in NCI-H295 cells is androgen synthesis, with formation of dehydroepiandrosterone, androstenedione, testotesterone, and at least three sulfated androgens, as well as estrogens. In addition, formation of cortisol, corticosterone, aldosterone, and 11 beta-hydroxyandrostenidione indicated the presence of 11 beta-hydroxylase. Thus, multiple pathways of steroidogenesis are expressed by NCI-H295 cells, including formation of corticosteroids, mineralocorticoids, androgens, and estrogens. Our findings indicate the presence in NCI-H295 cells of all of the major adrenocortical enzyme systems, including 11 beta-hydroxylase, desmolase, 21 alpha-hydroxylase, 17 alpha-hydroxylase, 18-hydroxylase, lyase, sulfokinase, and aromatase. The NCI-H295 cell line should prove of value in studying the regulation, metabolic pathways, and enzymes involved in steroid formation and secretion. In addition, it may provide insights into the biology and treatment of adrenocortical carcinoma.

Ecological correlation between arsenic level in well water and age-adjusted mortality from malignant neoplasms.

Abstract: A significant dose-response relation between ingested arsenic and several cancers has recently been reported in four townships of the endemic area of blackfoot disease, a unique peripheral artery disease related to the chronic arsenic exposure in southwestern Taiwan. This study was carried out to examine ecological correlations between arsenic level of well water and mortality from various malignant neoplasms in 314 precincts and townships of Taiwan. The arsenic content in water of 83,656 wells was determined by a standard mercuric bromide stain method from 1974 to 1976, while mortality rates of 21 malignant neoplasms among residents in study precincts and townships from 1972 to 1983 were standardized to the world population in 1976. A significant association with the arsenic level in well water was observed for cancers of the liver, nasal cavity, lung, skin, bladder and kidney in both males and females as well as for the prostate cancer in males. These associations remained significant after adjusting for indices of urbanization and industrialization through multiple regression analyses. The multivariate-adjusted regression coefficient indicating an increase in age-adjusted mortality per 100,000 person-years for every 0.1 ppm increase in arsenic level of well water was 6.8 and 2.0, 0.7 and 0.4, 5.3 and 5.3, 0.9 and 1.0, 3.9 and 4.2, as well as 1.1 and 1.7, respectively, in males and females for cancers of the liver, nasal cavity, lung, skin, bladder and kidney. The multivariate-adjusted regression coefficient for the prostate cancer was 0.5. These weighted regression coefficients were found to increase or remain unchanged in further analyses in which only 170 southwestern townships were included.

Growth Adaptation to Methotrexate of HT-29 Human Colon Carcinoma Cells Is Associated with Their Ability to Differentiate into Columnar Absorptive and Mucus-secreting Cells

Abstract: The purpose of this work was to investigate whether the phenomenon of metabolic adaptation of HT-29 cells to glucose deprivation and subsequent emergence of differentiated subpopulations (A. Zweibaum et al., J. Cell. Physiol., 122: 21–29, 1985) also applies to anticancer drugs that act at a metabolic level like methotrexate (MTX). Stepwise adaptation of exponentially growing HT-29 cells to increasing concentrations of MTX (10-7, 10-6, and 10-5 mol) results, after a phase of high mortality, in the emergence of subpopulations with stable growth rates and curves close to those of untreated control cells. In contrast to control cells which are heterogenous and contain, after confluency, only a small proportion of differentiated cell types (less than 4%), postconfluent cultures of MTX-adapted cells are totally differentiated. Cells adapted to 10-7m MTX form a mixed population of columnar absorptive and mucus cells; at higher concentrations cells are almost exclusively of the mucus-secreting type. All cells, whether mucus-secreting or not, develop an apical brush border which strongly expresses dipeptidylpeptidase IV, carcinoembryonic antigen, and villin. These differentiation features, which resemble those of fetal colon, are associated with decreased rates of glucose consumption and lactic acid production. Both differentiation characteristics and metabolic changes are stably maintained when the cells are subcultured in the absence of the drug. Like the original population, MTX-adapted cells are tumorigenic in nude mice. We propose that cells which are able to differentiate and which are the origin of the small proportion of differentiated cell types found in postconfluent cultures of the original cell line possess an advantage which allows them to be adaptable to “metabolic stress” conditions.

Boron Neutron Capture Therapy of Cancer

Abstract: The theoretical advantage of BCNT is that it is a two component or binary system, consisting of 10 B and thermal neutrons, which when combined together generate high LET radiation capable of selectively destroying tumor cells without significant damage to normal tissues. In order for BCNT to succeed a critical amount of 10 B and a sufficient number of thermal neutrons must be delivered to individual tumor cells

Characterization and Anticancer Activity of the Micelle-forming Polymeric Anticancer Drug Adriamycin-conjugated Poly(ethylene glycol)-Poly(aspartic acid) Block Copolymer

Abstract: Adriamycin (ADR), an anthracycline anticancer drug, was bound to the poly(aspartic acid) chain of poly(ethylene glycol)-poly(aspartic acid) block copolymer by amide bond formation between an amino group of Adriamycin and the carboxyl groups of the poly(aspartic acid) chain The polymeric drug thus obtained was observed to form a micelle structure possessing diameter of approximately 50 nm, with a narrow distribution, in phosphate-buffered saline and to show excellent water solubility despite a large amount of ADR introduction Further, it was able to be stored in lyophilized form without losing its water solubility in the redissolving procedure Increased stability of the bound Adriamycin molecules in phosphate-buffered saline and elimination of binding affinity for bovine serum albumin due to the micelle formation were further advantages of this polymeric drug In vivo high anticancer activity of this micelle-forming polymeric drug against P 388 mouse leukemia was obtained with less body weight loss than that seen with free ADR, due to low toxicity as compared with free ADR

Independent Expression and Cellular Processing of Mr 72,000 Type IV Collagenase and Interstitial Collagenase in Human Tumorigenic Cell Lines

Abstract: The regulation of M r 72,000 type IV collagenase and interstitial collagenase expression was studied in vitro . Three tumorigenic human cell lines were used, together with human fetal lung fibroblasts as a nontumorigenic control. M r 72,000 type IV collagenase was expressed constitutively by all four cell lines, whereas only A2058 melanoma cells exhibited constitutive expression of interstitial collagenase. Treatment of cells with transforming growth factor β1 (TGF-β1) and 12- O -tetradecanoylphorbol-13-acetate (TPA) revealed an opposite pattern of regulation of these two metalloproteinases. Specifically, TPA increased interstitial collagenase mRNA levels in each cell line and decreased type IV collagenase mRNA levels in control fibroblasts and the tumorigenic cell lines, HT-1080 and A2058. TGF-β1 treatment increased type IV collagenase mRNA levels in each cell line and decreased interstitial collagenase mRNA levels in A2058 melanoma cells. Interstitial collagenase mRNA induction was accompanied in all cell lines by elevated interstitial procollagenase in the conditioned medium, as detected by zymography. Changes in M r 72,000 type IV collagenase expression revealed a more complex pattern of regulation. TPA and TGF-β1 treatment of HT-1080 cells resulted in the appearance of two bands of gelatinolytic activity with a molecular weight of approximately 62,000 and 59,000. The M r 62,000 species was also induced by TGF-β1 treatment of A2058 cells. Addition of affinity-purified radiolabeled M r 72,000 type IV procollagenase to TPA-treated HT-1080 cells demonstrated that both species were products of the M r 72,000 proenzyme and that exogenous proenzyme could be processed by these cells. Western blot analysis with specific antipeptide antibodies revealed that both the M r 62,000 and 59,000 species were derived from the M r 72,000 proenzyme by amino-terminal cleavage. There was no evidence for cellular processing of either interstitial procollagenase or the M r 92,000 type IV procollagenase. These results demonstrate that the M r 72,000 type IV collagenase is under the control of different regulatory elements from interstitial collagenase, at the level of both mRNA expression and cellular processing, and that this processing appears to be the result of a phorbol ester and TGF-β1-inducible cellular activation mechanism. The ratio of active enzyme species to latent M r 72,000 proenzyme may provide a better correlation with invasive potential than overall levels of this widely expressed metalloproteinase.