Showing papers in "Cancer Research in 1996"

Association between Tumor Hypoxia and Malignant Progression in Advanced Cancer of the Uterine Cervix

Abstract: Experimental tumors contain a significant fraction of microregions that are chronically or transiently hypoxic. Experimental evidence showing that hypoxia (and subsequent reoxyenation) may have a profound impact on malignant progression and on responsiveness to therapy is growing. The clinical relevance of tumor oxygenation in human solid malignancies is under investigation. We have developed and validated a clinically applicable method for measurement of tumor oxygenation in locally advanced cancer of the uterine cervix using a computerized polarographic electrode system. Applying this procedure in patients with cervical cancers ≥3 cm in diameter, who gave informed consent, we have been studying the clinical relevance of tumor oxygenation prospectively since 1989. As of June 1995, 103 patients with advanced cancers of the uterine cervix [Federation Internationale des Gynaecologistes et Obstetristes (FIGO) stages Ib, bulky ( n = 13), IIa and IIb ( n = 51), IIIa and IIIb ( n = 34), and IVa and IVb ( n = 5)] had entered the study. Fifty % of the patients had carcinomas with median pO 2 readings hypoxic tumors. Tumor oxygenation was found to be independent of various patient demographics and also of pretreatment tumor characteristics, such as clinical tumor stage and size, histological type, and differentiation. However, histopathological examination of the surgical specimens following radical tumor resection in 47 patients showed that low-pO 2 tumors exhibited larger tumor extensions and more frequent (occult) parametrial spread, as well as lymph-vascular space involvement, compared to well-oxygenated tumors of similar clinical stage and size. Forty-two patients completing primary radiation therapy and 47 patients who underwent radical surgery were analyzed for treatment outcome after a median observation period of 28 months (range, 3–76 months). Patients with hypoxic tumors had significantly worse disease-free and overall survival probabilities compared to patients with nonhypoxic tumors. Cox regession analysis identified tumor oxygenation and FIGO stage as the most important independent prognostic factors. The poorer outcome of the patients with hypoxic tumors was mainly due to locoregional failures with and without distant metastases, irrespective of whether surgery or radiation was applied as primary treatment. Tumor oxygenation as measured with a standardized polarographic method proved to be a powerful new pretherapeutic prognostic parameter providing important information on malignant progression in terms of extracervical tumor spread and radioresistance in advanced cervical cancers.

Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma.

Abstract: This study was performed to explore the relationship between tumor oxygenation and treatment outcome in human soft tissue sarcoma. Twenty-two patients with nonmestastatic, high-grade, soft tissue sarcomas underwent preoperative irradiation and hyperthermia and pretreatment measurement of tumor oxygenation. The 18-month actuarial disease-free survival was 70% for patients with tumor median oxygen pressure (pO2) values of >10 mm Hg but only 35% for those with median pO2 values of <10 mm Hg (P=0.01). There were eight treatment failures; the first site of recurrence was lung in all patients. Median pO2 was 7.5 mm Hg for metastasizing tumors versus 20 mm Hg for nonmetastasizing tumors (P=0.03). Potential mechanisms and implications for clinical trial design are discussed.

Genetic progression model for head and neck cancer: Implications for field cancerization

Abstract: A genetic progression model of head and neck squamous cell carcinoma has not yet been elucidated, and the genetic basis for “field cancerization” of the aerodigestive tract has also remained obscure. Eighty-seven lesions of the head and neck, including preinvasive lesions and benign lesions associated with carcinogen exposure, were tested using microsatellite analysis for allelic loss at 10 major chromosomal loci which have been defined previously. The spectrum of chromosomal loss progressively increased at each histopathological step from benign hyperplasia to dysplasia to carcinoma in situ to invasive cancer. Adjacent areas of tissue with different histopathological appearance shared common genetic changes, but the more histopathologically advanced areas exhibited additional genetic alterations. Abnormal mucosal cells surrounding preinvasive and microinvasive lesions shared common genetic alterations with those lesions and thus appear to arise from a single progenitor clone. Based on these findings, the local clinical phenomenon of field cancerization seems to involve the expansion and migration of clonally related preneoplastic cells.

Association of Macrophage Infiltration with Angiogenesis and Prognosis in Invasive Breast Carcinoma

Abstract: Angiogenesis is a key process in tumor growth and metastasis and is a major independent prognostic factor in breast cancer A range of cytokines stimulate the tumor neovasculature, and tumor-associated macrophages have been shown recently to produce several important angiogenic factors We have quantified macrophage infiltration using Chalkley count morphometry in a series of invasive breast carcinomas to investigate the relationship between tumor-associated macrophage infiltration and tumor angiogenesis, and prognosis There was a significant positive correlation between high vascular grade and increased macrophage index (P = 003), and a strong relationship was observed between increased macrophage counts and reduced relapse-free survival (P = 0006) and reduced overall survival (P = 0004) as an independent prognostic variable These data indicate a role for macrophages in angiogenesis and prognosis in breast cancer and that this cell type may represent an important target for immunoinhibitory therapy in breast cancer

Inhibition of the Abl Protein-Tyrosine Kinase in Vitro and in Vivo by a 2-Phenylaminopyrimidine Derivative

Abstract: Oncogenic activation of Abl proteins due to structural modifications can occur as a result of viral transduction or chromosomal translocation The tyrosine protein kinase activity of oncogenic Abl proteins is known to be essential for their transforming activity Therefore, we have attempted to identify selective inhibitors of the Abl tyrosine protein kinase Herein we describe an inhibitor (CGP 57148) of the Abl and platelet-derived growth factor (PDGF) receptor protein-tyrosine kinases from the 2-phenylaminopyrimidine class, which is highly active in vitro and in vivo Submicromolar concentrations of the compound inhibited both v-Abl and PDGF receptor autophosphorylation and PDGF-induced c-fos mRNA expression selectively in intact cells In contrast, ligand-induced growth factor receptor autophosphorylation in response to epidermal growth factor (EGF), insulin-like growth factor-I, and insulin showed no or weak inhibition by high concentrations of CGP 57148 c-fos mRNA expression induced by EGF, fibroblast growth factor, or phorbol ester was also insensitive to inhibition by CGP 57148 In antiproliferative assays, the compound was more than 30-100-fold more potent in inhibiting growth of v-abl-transformed PB-3c cells and v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/MK cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line Furthermore, anchorage-independent growth of v-abl- and v-sis-transformed BALB/c 3T3 cells was inhibited potently by CGP 57148 When tested in vivo, CGP 57148 showed antitumor activity at tolerated doses against tumorigenic v-abl- and v-sis-transformed BALB/c 3T3 cells In contrast, CGP 57148 had no antitumor activity when tested using src-transformed BALB/c 3T3 cells These findings suggest that CGP 57148 may have therapeutic potential for the treatment of diseases that involve abnormal cellular proliferation induced by Abl protein-tyrosine kinase deregulation or PDGF receptor activation

Cellular pH gradient in tumor versus normal tissue : Potential exploitation for the treatment of cancer

Abstract: Although limited data exist, electrode-measured pH values of human tumors and adjacent normal tissues, which are concurrently obtained by the same investigator in the same patient, consistently show that the electrode pH (believed to primarily represent tissue extracellular pH) is substantially and consistently lower in the tumor than in normal tissue. In contrast, the 31P-magnetic resonance spectroscopy estimated that intracellular pH is essentially identical or slightly more basic in tumor compared to normal tissue. As a consequence, the cellular pH gradient is substantially reduced or reversed in tumor compared to normal tissue: in normal tissue the extracellular pH is relatively basic, and in tumor tissue the magnitude of the pH gradient is reduced or reversed. The difference provides an exploitable avenue for the treatment of cancer. The extent to which drugs exhibiting weakly acid or basic properties are ionized is strongly dependent on the pH of their milieu. Weakly acidic drugs which are relatively lipid soluble in their nonionized state may diffuse freely across the cell membrane and, upon entering a relatively basic intracellular compartment, become trapped and accumulate within a cell, leading to substantial differences in the intracellular/extracellular drug distribution between tumor and normal tissue for drugs exhibiting appropriate pKas.

Treatment of Established Tumors with a Novel Vaccine That Enhances Major Histocompatibility Class II Presentation of Tumor Antigen

Abstract: Presentation of antigenic peptides by MHC class II molecules to CD4+ T cells is critical to the generation of antitumor immunity. In an attempt to enhance MHC class II antigen processing, we linked the sorting signals of the lysosome-associated membrane protein (LAMP-1) to the cytoplasmic/nuclear human papilloma virus (HPV-16) E7 antigen, creating a chimera (Sig/E7/LAMP-1). Previously, we found that expression of this chimera in vitro and in vivo with a recombinant vaccinia vector targeted E7 to endosomal and lysosomal compartments and enhanced MHC class II presentation to CD4+ T cells compared to vaccinia expressing wild-type E7. In the current study, we tested these recombinant vaccinia for in vivo protection against an E7+ tumor, TC-1, which was derived from primary epithelial cells of C57BL/6 mice cotransformed with HPV-16 E6 and E7 and c-Ha-ras oncogenes. All mice vaccinated with 1 x 10(7) plaque-forming units of wild-type E7-vaccinia showed progressive tumor growth when challenged with a tumorigenic dose of TC-1 tumor cells; in contrast, 80% of mice vaccinated with the chimeric Sig/E7/LAMP1 vaccinia remained tumor free 3 months after tumor injection. Furthermore, treatment with the Sig/E7/LAMP-1 vaccinia vaccine cured mice with small established TC-1 tumors, whereas the wild-type E7-vaccinia showed no effect on this established tumor burden. These findings point out the therapeutic limitations of recombinant vaccinia expressing unmodified tumor antigens. Further, they demonstrate that modifications that reroute a cytosolic tumor antigen to the endosomal/lysosomal compartment can profoundly improve the in vivo therapeutic potency of recombinant vaccines.

Tumor Malignancy Defined by Aberrant Glycosylation and Sphingo(glyco)lipid Metabolism

Abstract: Aberrant glycosylation expressed in glycosphingolipids and glycoproteins in tumor cells has been implicated as an essential mechanism in defining stage, direction, and fate of tumor progression This general concept is supported by results from three lines of study: (a) Numerous clinicopathological studies have shown a clear correlation between aberrant glycosylation status of primary tumor and invasive/metastatic potential of human cancer as reflected by 5- or 10-year survival rates of patients (b) Carbohydrates expressed in tumor cells are either adhesion molecules per se or modulate adhesion receptor function Some are directly involved in cell adhesion They are recognized by selectins or other carbohydrate-binding proteins or by complementary carbohydrates (through carbohydrate-carbohydrate interaction) N- or O-glycosylation of functionally important membrane components may alter tumor cell adhesion or motility in a direction that either promotes or inhibits invasion and metastasis Examples of such receptors are E-cadherin, integrins, immunoglobulin family receptors (eg, CD44), and lysosome-associated membrane protein (c) Gangliosides and sphingolipids modulate transmembrane signaling essential for tumor cell growth, invasion, and metastasis The transducer molecules susceptible to gangliosides and sphingolipids include integrin receptors, tyrosine kinase-linked growth factor receptors, protein kinase C, and G-protein-linked receptor affecting protein kinase A Some glycosphingolipids (eg, Gb3Cer, Le(y), ceramide, and sphingosine induce tumor cell differentiation and subsequent apoptosis Shedded gangliosides may block immunogenicity of tumor cells, providing conditions favorable for "escape" from immunological suppression of tumor growth by the host Various reagents that block carbohydrate-mediated tumor cell adhesion or block glycosylation processing have been shown to inhibit tumor cell metastasis This provides the basis for further development of "anti-adhesion therapy" Ganglioside analogues and sphingolipid analogues that inhibit protein kinase C and receptor-associated tyrosine kinase have been applied for inhibition of metastasis A crucial mechanism for inhibition of metastasis by these reagents may involve blocking of transmembrane signaling for expression of P- and E-selectin This provides the basis for development of "ortho-signaling therapy"

E7 Protein of Human Papilloma Virus-16 Induces Degradation of Retinoblastoma Protein through the Ubiquitin-Proteasome Pathway

Abstract: Rb protein is a critical regulator of entry into the cell cycle, and loss of Rb function by deletions, mutations, or interaction with DNA viral oncoproteins leads to oncogenic transformation. We have shown that the human papilloma virus (HPV)-16 E7 gene is sufficient to induce the immortalization of mammary epithelial cells (MECs). Surprisingly, the steady-state level of Rb protein in these immortal cells was drastically decreased. Here, we used pulse-chase analysis to show that the in vivo loss of Rb protein in E7-immortalized MECs is a consequence of enhanced degradation. Expression of HPV16 E7 in a cell line with a temperature-sensitive mutation in the E1 enzyme of the ubiquitin pathway demonstrated that degradation of Rb was ubiquitin dependent. Treatment of E7-immortalized MECs with aldehyde inhibitors of proteasome-associated proteases led to a marked stabilization of Rb protein, particularly the hypophosphorylated form. Taken together, our results provide evidence for HPV-16 E7-induced enhanced degradation of Rb protein via a ubiquitin-proteasome pathway and suggest a second mechanism of oncogenic transformation by E7, in addition to its previously identified ability to sequester Rb from E2F. Our analyses also show that normal Rb levels are regulated by the ubiquitin-proteasome degradation pathway.

Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1

Abstract: A human cytochrome P-450 (P450) 1B1 cDNA was expressed in Saccharomyces cerevisiae , and the microsomes containing P450 1B1 were used to examine the selectivity of this enzyme in the activation of a variety of environmental carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strains, using the SOS response as an end point of DNA damage. We also determined and compared these activities of P450 1B1 with those catalyzed by recombinant human P450s 1A1 and 1A2, which were purified from membranes of Escherichia coli . The carcinogenic chemicals tested included 27 polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, 17 heterocyclic and aryl amines and aminoazo dyes, three mycotoxins, two nitroaromatic hydrocarbons, N -nitrosodimethylamine, vinyl carbamate, and acrylonitrile. Among the three P450 enzymes examined here, P450 1B1 was found to have the highest catalytic activities for the activation of 11,12-dihydroxy-11,12-dihydrodibenzo[ a,l ]pyrene, 1,2-dihydroxy-1,2-dihydro-5-methylchrysene, (+)-7,8-dihydroxy-7,8-dihydrobenzo[ a ]pyrene, 11,12-dihydroxy-11,12-dihydrobenzo[ g ]chrysene, 3,4-dihydroxy-3,4-dihydrobenzo[ c ]phenanthrene, 3-amino-1,4-dimethyl-5 H -pyrido[4,3- b ]indole, 2-aminoanthracene, 3-methoxy-4-aminoazobenzene, and 2-nitropyrene. P450 1B1 also catalyzed the activation of 2-amino-3,5-dimethylimidazo[4,5- f ]quinoline, 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline, 2-amino-3-methylimidazo[4,5- f ]quinoline, 2-aminofluorene, 6-aminochrysene and its 1,2-dihydrodiol, (-)-7,8-dihydroxy-7,8-dihydrobenzo[ a ]pyrene, 1,2-dihydroxy-1,2-dihydrochrysene, 1,2-dihydroxy-1,2-dihydro-5,6-dimethylchrysene, 2,3-dihydroxy-2,3-dihydrofluoranthene, 3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[ a ]anthracene, and 6-nitrochrysene to appreciable extents. However, P450 1B1 did not produce genotoxic products from benzo[ a ]pyrene, trans -3,4-dihydroxy-3,4-dihydrobenzo[ a ]anthracene, trans -8,9-dihydroxy-8,9-dihydrobenzo[ a ]anthracene, 7,12-dimethylbenz[ a ]anthracene and its cis -5,6-dihydrodiol, 5-methylchrysene, 11,12-dihydroxy-11,12-dihydro-3-methylcholanthrene, 1,2-dihydroxy-1,2-dihydro-6-methylchrysene, benzo[ c ]phenanthrene, 2-amino-6-methyldipyrido[1,2- a :3′,2′- d ]imidazole, 2-acetylaminofluorene, benzidine, 2-naphthylamine, aflatoxin B 1 , aflatoxin G 1 , sterigmatocystin, N -nitrosodimethylamine, vinyl carbamate, or acrylonitrile in this assay system. P450 1B1 is expressed constitutively in extrahepatic organs, including fetal tissue samples, and is highly inducible in various organs by 2,3,7,8-tetrachlorodibenzo- p -dioxin and related compounds in experimental animal models. Thus, activation of procarcinogens by P450 1B1 may contribute to human tumors of extrahepatic origin.

Mitotic Block Induced in HeLa Cells by Low Concentrations of Paclitaxel (Taxol) Results in Abnormal Mitotic Exit and Apoptotic Cell Death

Abstract: Paclitaxel at low concentrations (10 nm for 20 h) induces ∼90% mitotic block at the metaphase/anaphase transition in HeLa cells, apparently by suppressing dynamics of spindle microtubules (M. A. Jordan et al., Proc. Natl. Acad. Sci. USA, 90: 9552–9556, 1993). It is not known, however, whether inhibition of mitosis by such low paclitaxel concentrations results in cell death. In the present work, we found that after removal of pacli-taxel (10 nm-1 µm), blocked cells did not resume proliferation. Instead, cells exited mitosis abnormally within 24 h. They did not progress through anaphase or cytokinesis but entered an interphase-like state (chromatin decondensed, and an interphase-like microtubule array and nuclear membranes reformed). Many cells (≥55%) contained multiple nuclei. Additional DNA synthesis and polyploidy did not occur. DNA degradation into nucleosome-sized fragments characteristic of apoptosis began during drug incubation and increased after drug removal. Cells died within 48–72 h. Incubation with paclitaxel (10 nm for 20 h) resulted in high intracellular drug accumulation (8.3 µm) and little efflux after paclitaxel removal; intracellular retention of paclitaxel may contribute to its efficacy. The results support the hypothesis that the most potent chemotherapeutic mechanism of paclitaxel is kinetic stabilization of spindle microtubule dynamics.

DPC4 Gene in Various Tumor Types

Abstract: We recently identified a novel tumor-suppressor gene, DPC4 , at chromosome 18q21.1 and found that both alleles of DPC4 were inactivated in nearly one-half of the pancreatic carcinomas. Here, we analyzed 338 tumors, originating from 12 distinct anatomic sites, for alterations in the DPC4 gene. Sixty-four specimens were selected for the presence of the allelic loss of 18q and were further analyzed for DPC4 sequence alterations. An alteration of the DPC4 gene sequence was identified in one of eight breast carcinomas and one of eight ovarian carcinomas. These results indicate that whereas DPC4 inactivation is prevalent in pancreatic carcinoma (48%), it is distinctly uncommon (<10%) in the other tumor types examined. The tissue restriction of alterations in DPC4 , as in many other tumor-suppressor genes, emphasizes the complexity of rate-limiting checkpoints in human tumorigenesis.

Taxol induces bcl-2 phosphorylation and death of prostate cancer cells.

Abstract: Treatment of prostate cancer cell lines expressing bcl-2 with taxol induces bcl-2 phosphorylation and programmed cell death, whereas treatment of bcl-2-negative prostate cancer cells with taxol does not induce apoptosis. bcl-2 phosphorylation seems to inhibit its binding to bax since less bax was observed in immunocomplex with bcl-2 in taxol-treated cancer cells. These findings support the use of the anticancer drug taxol for the treatment of bcl-2-positive prostate cancers and other bcl-2-positive malignancies, such as follicular lymphoma.

In Vivo Ubiquitination and Proteasome-mediated Degradation of p53

Abstract: The levels of the tumor suppressor protein p53 are generally quite low in normal cells, due in part to its rapid turnover. Previous studies have implicated ubiquitin-dependent proteolysis in the turnover of wild-type p53 but have not established whether or not p53 is itself a substrate of the ubiquitin system. In this study, inhibitors of the 26S proteasome have been used to further explore the role of ubiquitin proteolysis in regulating p53 turnover. Increased levels of the tumor suppressor protein p53 were observed in normal cells, as well as in cells expressing the human papillomavirus 16 E6 oncoprotein, on exposure of the cells to proteasome inhibitors. Pulse-chase experiments indicated that the increased p53 levels resulted from stabilization of the protein. Furthermore, ubiquitin-p53 conjugates were detected in untreated as well as gamma-irradiated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of p53. Increased levels of the cyclin:cyclin-dependent kinase inhibitor p21, a downstream effector of p53 function, were also observed in proteasome inhibitor-treated cells, and this increase was due in part to an increase in p2l mRNA.

Clinical Experiences with Magnetic Drug Targeting: A Phase I Study with 4′-Epidoxorubicin in 14 Patients with Advanced Solid Tumors

Abstract: Anticancer drugs reversibly bound to magnetic fluids (ferrofluids) could be concentrated in locally advanced tumors by magnetic fields that are arranged at the tumor surface outside of the organism. If certain requirements are met, systemic toxicity might be minimized, and local tumor efficacy might be increased. We have conducted a Phase I clinical trial using this approach in patients with advanced and unsuccessfully pretreated cancers or sarcomas. Nine such patients received two treatment courses, 3 patients received one course, and 2 patients received three courses of magnetic drug targeting consisting of the infusion of epirubicin in increasing doses (from 5 to 100 mg/m2) that had been chemically bound to a magnetic fluid and the application of magnetic fields to the tumors for 60–120 min. In 2 of 14 patients, the same dose of epirubicin not bound to a magnetic fluid was administered systemically 3 weeks after drug targeting for intraindividual comparisons. Magnetic drug targeting with epirubicin was well tolerated. In one case, a planned second treatment was withdrawn, because of an episode of chills 130 min after infusion of the magentic drug. Two patients received a third treatment because of good responses after the first two therapies. Based on magnetic resonance tomographic techniques, pharmacokinetics, and the histological detection of magnetites, it was shown that the ferrofluid could be successfully directed to the tumors in about one-half of the patients. Organ toxicity did not increase with the treatment, but epirubicin-associated toxicity appeared at doses greater than 50 mg/m2. Although treatment with magnetic drug targeting seems safe, improvements are necessary to make it more effective and independent of patient- or disease-related problems. A study design to compare conventional treatments with the new treatment form within one patient seems crucial to eliminate interindividual differences.

Germline BRCA2 Gene Mutations in Patients with Apparently Sporadic Pancreatic Carcinomas

Abstract: Germline mutations in BRCA2 predispose carriers to the development of breast, ovarian, and a variety of other cancers. The original localization of the BRCA2 gene was aided by its homozygous deletion in a pancreatic carcinoma; indeed, an excess of pancreatic carcinoma has been seen in some BRCA2 cancer families. To determine the involvement of BRCA2 in pancreatic carcinomas, we screened for BRCA2 alterations in an unselected panel of 41 adenocarcinomas of the pancreas (30 pancreatic adenocarcinoma xenografts and 11 pancreatic cancer cell lines). Of the 15 (27%) that had allelic loss at the BRCA2 locus, 4 (9.8%) had abnormalities in the second allele upon screening of the entire BRCA2 gene by in vitro synthesized protein assay. Three of the four mutations were considered germline in origin (7.3% overall; two were confirmed in normal tissue, and one was the 6174delT mutation from the pancreatic cancer cell line CAPAN-1, for which normal tissue was unavailable). The identification of two 6174delT mutations in this series prompted us to evaluate the prevalence of this mutation in an overlapping consecutive series of 245 patients who underwent pancreatoduodenectomy for adenocarcinoma of the pancreas. Sequence analysis of this limited region of the gene identified two additional mutations: ( a ) one additional germline 6174delT mutation (2 of 245, 0.8% overall); and ( b ) a second nearby germline 6158insT mutation. One of the patients with a germline mutation had a single relative with breast cancer, and another had a single relative with prostate cancer. None had a family history of pancreatic cancer. The incidence of germline BRCA2 mutations in apparently sporadic pancreatic cancer may be at least as high as in breast or ovarian cancer. Our results suggest that some familial risks for carcinoma will be evident only through a population-based application of gene screening techniques because a low disease penetrance of the germline mutations in some families often evades clinical suspicion.

The role of DNA mismatch repair in platinum drug resistance.

Abstract: Loss of DNA mismatch repair occurs in many types of tumors. The effect of the loss of DNA mismatch repair activity on sensitivity to cisplatin and a panel of analogues was tested using two pairs of cell lines proficient or deficient in this function. HCT116+ch2, a human colon cancer cell line deficient in hMLH1, was 2.1-fold resistant to cisplatin and 1.3-fold resistant to carboplatin when compared to a subline complemented with chromosome 3 expressing a wild-type copy of hMLH1. Likewise, the human endometrial cancer cell line HEC59, which is deficient in hMSH2, was 1.8-fold resistant to cisplatin and 1.5-fold resistant to carboplatin when compared to a subline complemented with chromosome 2 with a wild-type hMSH2. In contrast to cisplatin and carboplatin, which form the same types of adducts in DNA, there was no difference in sensitivity between the DNA mismatch repair-proficient and -deficient cell lines for oxaliplatin, tetraplatin, transplatin, JM335, or JM216. The formation of protein-DNA complexes that contained hMSH2 and hMLH1 was documented by mobility shift assay when nuclear extracts were incubated with DNA platinated with cisplatin but not with oxaliplatin. These results demonstrate a correlation between failure of the DNA mismatch repair proteins to recognize the platinum adduct and low-level resistance, suggesting a role for the DNA mismatch repair system in generating signals that contribute to the generation of apoptotic activity. They also identify the use of drugs whose adducts are not recognized as a strategy for circumventing resistance due to loss of DNA mismatch repair.

Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B

Abstract: The recently identified cyclin-dependent kinase inhibitor p15INK4B is localized to a region on chromosome 9p21 frequently deleted in human tumors. Previous evidence has pointed to a related gene, p16INK4A, as the principal target of this deletion. We report that in gliomas and, to a striking degree, in leukemias, the p15 gene is commonly inactivated in association with promoter region hypermethylation involving multiple sites in a 5'-CpG island. In some gliomas and all of the primary leukemias, this event occurs without alteration of the adjacent gene, p16INK4A. In other tumors, including lung, head and neck, breast, prostate, and colon cancer, inactivation of p15INK4B occurs only rarely and only with concomitant inactivation of p16. Aberrant methylation of p15INK4B is associated with transcriptional loss of this gene. Treatment with the demethylating agent 5-aza-2'-deoxycytidine leads to re-expression of p15 mRNA. In selected leukemia cell lines, p15 inactivation correlates with known resistance to the growth-suppressive effects of transforming growth factor-beta. These results suggest that p15INK4B is inactivated selectively in leukemias and gliomas and seems to constitute an important tumor suppressor gene loss in these neoplasms.

Metastatic Prostate Cancer in a Transgenic Mouse

Abstract: We have previously reported the development of a transgenic mouse model for prostate cancer derived from PB-Tag transgenic line 8247, henceforth designated the TRAMP (transgenic adenocarcinoma mouse prostate) model. We now describe the temporal and spatial consequences of transgene expression and report the identification and characterization of metastatic disease in the TRAMP model. TRAMP mice characteristically express the T antigen oncoprotein by 8 weeks of age and develop distinct pathology in the epithelium of the dorsolateral prostate by 10 weeks of age. Distant site metastases can be detected as early as 12 weeks of age. The common sites of metastases are the periaortic lymph nodes and lungs, with occasional metastases to the kidney, adrenal gland, and bone. By 28 weeks of age, 100% harbor metastatic prostate cancer in the lymph nodes or lungs. We have also demonstrated the loss of normal E-cadherin expression, as observed in human prostate cancer, as primary tumors become less differentiated and metastasize. The TRAMP model provides a consistent source of primary and metastatic tumors for histopathobiological and molecular analysis to further define the earliest molecular events involved in the genesis, progression, and metastasis of prostate cancer.

Cyclooxygenase-2 Overexpression and Tumor Formation Are Blocked by Sulindac in a Murine Model of Familial Adenomatous Polyposis

Abstract: Inducible cyclooxygenase (Cox-2), also known as prostaglandin H synthase 2 (PGH-2) is a key enzyme in the formation of prostaglandins and thromboxanes. Cox-2 is the product of an immediate-early gene that is expressed in response to growth factors, tumor promoters, or cytokines. Overexpression of Cox-2 is associated with both human colon cancers and suppression of apoptosis in cultured epithelia] cells, an activity that is reversed by the nonsteroidal anti-inflammatory drug, sulindac sulfide. To address the relationship between Cox-2, apoptosis, and tumor development in vivo, we studied C57BL/6J-Min/+(Min) mice, a strain containing a fully penetrant dominant mutation in the Apc gene, leading to the development of gastrointestinal adenomas by 110 days of age. Min mice were fed AIN-76A chow diet and given sulindac (0.5 +/- 0.1 mg/day) in drinking water. Control Min mice and homozygous C57BL/6J-+/+ normal littermates lacking the Apc mutation (+/+) were fed AIN-76A diet and given tap water to drink. At 110 days of age, all mice were sacrificed, and their intestinal tracts were examined. Control Min mice had 11.9 +/- 7.8 tumors per mouse compared to 0.1 +/- 0.1 tumors for sulindac-treated Min mice. As expected, +/+ littermates had no macroscopic tumors. Examination of histologically normal-appearing small bowel from Min animals revealed increased amounts of Cox-2 and prostaglandin E(2) compared to +/+ littermates. Using two different in situ techniques, terminal transferase-mediated dUTP nick end labeling and a direct immunoperoxidase method, Min animals also demonstrated a 27-47% decrease in enterocyte apoptosis compared to +/+ animals. Treatment with sulindac not only inhibited tumor formation but decreased small bowel Cox-2 and prostaglandin E(2) to baseline and restored normal levels of apoptosis. These data suggest that overexpression of Cox-2 is associated with tumorigenesis in the gastrointestinal epithelium, and that both are inhibited by sulindac administration.

Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump.

Abstract: Previous studies have identified the ATP-dependent export of glutathione conjugates as a physiological function of the multidrug resistance protein (MRP). The involvement of MRP in the transport of endogenous and xenobiotic conjugates was investigated further using membrane vesicles from MRP -transfected HeLa cells. The ATP-dependent transport of the glutathione conjugates [3H]leukotriene C4, S -(2,4-dinitrophenyl)-[3H]glutathione, and 3H-labeled oxidized glutathione was characterized by determination of the transport efficiency Vmax: K m amounting to 1031, 114, and 7.1 ml × mg protein-1 × min-1, respectively. Additional endogenous substrates for MRP-mediated transport included the steroid conjugate 17β-glucuronosyl [3H]estradiol and the bile salt conjugates [6α-14C]glucuronosylhyodeoxycholate and 3α-sulfatolithocholyl [3H]taurine. The K m value of MRP for 17β-glucuronosyl [3H]estradiol was 1.5 ± 0.3 µm, with a Vmax: K m ratio of 42 ml × mg protein-1 × min-1, and a K i value of 0.7 µm for the leukotriene receptor antagonist MK 571. MRP-mediated ATP-dependent transport was observed for the anticancer drug conjugates glucuronosyl [3H]etoposide and monochloro-mono[3H]glutathionyl melphalan, but not for unmodified [14C]doxorubicin, [3H]daunorubicin, or [3H]vinblastine. Our results establish that MRP functions as an ATP-dependent export pump not only for glutathione conjugates but also for glucuronidated and sulfated endogenous as well as exogenous compounds.

High Frequency of p16 (CDKN2/MTS-1/INK4A) Inactivation in Head and Neck Squamous Cell Carcinoma

Abstract: The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) can be inactivated by multiple genetic mechanisms. We analyzed 29 invasive primary head and neck squamous cell carcinomas (HNSCC) for p16 inactivation with immunohistochemistry utilizing a new monoclonal antibody (mAb), DCS-50. p16 staining of the primary lesions was correlated with genetic analysis including: (a) detailed microsatellite analysis of markers at the p16 locus to detect homozygous deletion; (b) sequence analysis of p16; and (c) Southern blot analysis to determine the methylation status of the 5' CpG island of p16. Twenty-four of 29 (83%) head and neck squamous cell carcinoma tumors displayed an absence of p16 nuclear staining using immunohistochemistry. Of these 24 tumors, we found that 16 (67%) harbored homozygous deletions, 5 (21%) were methylated, 1 displayed a rearrangement at the p16 locus, and 1 displayed a frameshift mutation in exon 1. These data suggest that: (a) inactivation of the p16 tumor suppressor gene is a frequent event in squamous cell carcinomas of the head and neck; (b) p16 is inactivated by several distinct and exclusive events including homozygous deletion, point mutation, and promoter methylation; and (c) immunohistochemical analysis for expression of the p16 gene product is an accurate and relatively simple method for evaluating p16 gene inactivation.

A Reaction-Diffusion Model of Cancer Invasion

Abstract: We present mathematical analyses, experimental data, and clinical observations which support our novel hypothesis that tumor-induced alteration of microenvironmental pH may provide a simple but complete mechanism for cancer invasion. A reaction-diffusion model describing the spatial distribution and temporal development of tumor tissue, normal tissue, and excess H+ ion concentration is presented. The model predicts a pH gradient extending from the tumor-host interface, which is confirmed by reanalysis of existing experimental data. Investigation of the structure and dynamics of the tumor-host interaction within the context of the model demonstrates a transition from benign to malignant growth analogous to the adenoma-carcinoma sequence. The effect of biological parameters critical to controlling this transition are supported by experimental and clinical observations. Tumor wave front velocities determined via a marginal stability analysis of the model equations are consistent with in vivo tumor growth rates. The model predicts a previously unrecognized hypocellular interstitial gap at the tumor-host interface which we demonstrate both in vivo and in vitro. A direct correlation between the interfacial morphology and tumor wave front velocity provides an explicit, testable, clinically important prediction.

Antioxidant Supplementation Decreases Oxidative DNA Damage in Human Lymphocytes

Abstract: The association between high intake of fruit and vegetables and low incidence of certain cancers is well established. Dietary antioxidants present in these foods are thought to decrease free radical attack on DNA and hence to protect against mutations that cause cancer, but this causal mechanism remains conjectural. We have adopted a molecular epidemiological approach to this question, based on a modified alkaline single-cell gel electrophoresis assay ("comet assay") which specifically detects oxidation of pyrimidines in the DNA of human lymphocytes. In a survey of men 50-59 years of age living in the northeast of Scotland, smokers initially showed significantly more base damage than nonsmokers. Correlations between oxidative base damage and plasma concentrations of various antioxidants were generally negative but not statistically significant. Supplementation of the diet for 20 weeks with vitamin C (100 mg/day), vitamin E (280 mg/day), and beta-carotene (25 mg/day) resulted in a highly significant (P < 0.002) decrease in endogenous oxidative base damage in the lymphocyte DNA of both smokers and nonsmokers. In addition, lymphocytes of antioxidant-supplemented subjects showed an increased resistance to oxidative damage when challenged in vitro with H2O2. These findings strongly support the hypothesis that fruit and vegetables exert a cancer-protective effect via a decrease in oxidative damage to DNA.

Flavopiridol Induces G1 Arrest with Inhibition of Cyclin-dependent Kinase (CDK) 2 and CDK4 in Human Breast Carcinoma Cells

Abstract: Flavopiridol (L86-8275), a N-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle progression in either G1 or G2 and is a potent cyclin-dependent kinase (CDK) 1 inhibitor. In this study, we used MCF-7 breast carcinoma cells that are wild type for p53 and pRb positive and contain CDK4-cyclin D1 and MDA-MB-468 breast carcinoma cells that are mutant p53, pRb negative, and lack CDK4-cyclin D1 to investigate the G1 arrest produced by Flavopiridol. Recombinant CDK4-cyclin D1 was inhibited potently by Flavopiridol (Kiapp, 65 nM), competitive with respect to ATP. Surprisingly, CDK4 immunoprecipitates derived from Flavopiridol-treated MCF-7 cells (3 h, 300 nM Flavonolpiridol) had an approximately 3-fold increased kinase activity compared with untreated cells. Cyclin D and CDK4 levels were not different at 3 hr, but cyclin D levels and CDK4 kinase activity decreased thereafter. The phosphorylation state of pRb was shifted from hypercoincident to hypocoincident with the development of G1 arrest. Asynchronous MDA-MB-468 cells were inhibited in cell cycle progression at both G1 and G2 by Flavopiridol. Flavopiridol inhibited the in vitro kinase activity of CDK2 using an immune complex kinase assay (IC50, 100 nM at 400 microM ATP). Immunoprecipitated CDK2 kinase activity from either MCF-7 or MDA-MB-468 cells exposed to Flavopiridol (300 nM) for increasing time showed an initial increased activity (approximately 1.5-fold at 3 h) compared with untreated cells, followed by a loss of kinase activity to immeasurable levels by 24 h. This increased immunoprecipitated kinase activity was dependent on the Flavopiridol concentration added to intact cells and was associated with a reduction of CDK2 tyrosine phosphorylation. Cyclin E and A levels were not altered to the same extent as cyclin D, and neither CDK4 nor CDK2 levels were changed in response to Flavopiridol. Inhibition of the CDK4 and/or CDK2 kinase activity by Flavopiridol can therefore account for the G1 arrest observed after exposure to Flavopiridol.

A Methylenetetrahydrofolate Reductase Polymorphism and the Risk of Colorectal Cancer

Abstract: We examined the relationship of a common polymorphism (667C→T) of the methylenetetrahydrofolate reductase (MTHFR) gene with the risk of colorectal cancer in a case-control study conducted in the Health Professionals Follow-up Study. MTHFR genotypes were ascertained from blood samples among 144 men previously diagnosed with colorectal cancer and 627 controls. The adjusted odds ratio (OR) for the MTHFR variant homozygous ( val/val ) genotype was 0.57 [95% confidence interval (CI), 0.30–1.06]. High dietary intake of methionine (OR, 0.27; 95% CI, 0.06–1.20) and low consumption of alcohol (OR, 0.11; 95% CI, 0.01–0.85) were associated with reduced incidence of colorectal cancer. Alcohol intake was a stronger risk factor among men with the val/val genotype ( P , trend = 0.01), and consumption of five or more alcoholic drinks per week abolished the reduced risk of colorectal cancer among val/val individuals ( P , interaction = 0.02). The inverse association of methionine with colorectal cancer risk was slightly stronger among individuals with the MTHFR val/val genotype. These data suggest that dietary methyl supply is particularly critical among MTHFR val/val individuals. When dietary methyl supply is high, MTHFR val/val individuals may be at reduced risk of colorectal cancer probably because higher levels of 5,10-methylenetetrahydrofolate may prevent imbalances of nucleotide pools during DNA synthesis. In contrast, when 5-methyltetrahydrofolate is depleted by alcohol consumption, val/val individuals may be less able to compensate, leading to potentially oncogenic alterations in DNA methylation.

Loss of DNA mismatch repair in acquired resistance to cisplatin.

Abstract: Selection of cells for resistance to cisplatin, a well-recognized mutagen, could result in mutations in genes involved in DNA mismatch repair and thereby to resistance to DNA-alkylating agents. Parental cells of the human ovarian adenocarcinoma cell line 2008 expressed hMLH1 when analyzed with immunoblot. One subline selected for resistance to cisplatin (2008/A) expressed no hMLH1, whereas another (2008/C13*5.25) expressed parental levels. Microsatellite instability was readily demonstrated in 2008/A cells but not in 2008 and in 2008/C13*5.25 cells. In addition, the 2008/A cells were 2-fold resistant to methyl-nitro-nitrosoguanidine and had a 65-fold elevated mutation rate at the HPRT locus as compared to 2008 cells, both of which are consistent with the loss of DNA mismatch repair in these cells. To determine whether the loss of DNA mismatch repair itself contributes to cisplatin resistance, studies were carried out in isogenic pairs of cell lines proficient or defective in this function. HCT116, a human colon cancer cell line deficient in hMLH1 function, was 2-fold resistant to cisplatin when compared to a subline complemented with chromosome 3 and expressing hMLH1. Similarly, the human endometrial cancer cell line HEC59, which expresses no hMSH2, was 2-fold resistant to cisplatin when compared to a subline complemented with chromosome 2 that expresses hMSH2. Therefore, the selection of cells for resistance to cisplatin can result in the loss of DNA mismatch repair, and loss of DNA mismatch repair in turn contributes to resistance to cisplatin.

Inducible Nitric Oxide Synthase, Nitrotyrosine, and Apoptosis in Helicobacter pylori Gastritis: Effect of Antibiotics and Antioxidants

Abstract: Helicobacter pylori infection is a known risk factor for gastric cancer. We hypothesized that H. pylori infection would lead to the sustained production of the reactive nitrogen species nitric oxide and peroxynitrite as part of the host immune response. We further hypothesized that H. pylori infection would lead to increased apoptosis of gastric epithelial cells, possibly in response to free radical-mediated DNA damage. Using immunohistochemistry, we stained and scored gastric antral biopsies from 84 Colombian patients with nonatrophic gastritis before and after treatment for H. pylori infection. We examined expression of inducible nitric oxide synthase (iNOS); nitrotyrosine, a marker for peroxynitrite; and DNA fragmentation, a marker for apoptosis. Patients were treated with triple therapy (amoxicillin, 500 mg three times a day for 2 weeks; metronidazole, 400 mg three times a day for 2 weeks; and bismuth subsalicylate, 262 mg four times a day for 2 weeks, followed by 262 mg every day for 4-12 months). Eradication of H. pylori infection resulted in a significant reduction in iNOS and nitrotyrosine staining and a marginally significant reduction in apoptosis. Dietary supplementation with beta-carotene (30 mg every day for 4-12 months) resulted in a significant decrease in iNOS staining. Supplementation with ascorbic acid (1 g twice a day for 4-12 months) led to a significant reduction in nitrotyrosine staining. In patients supplemented with either ascorbic acid or beta-carotene, there was a trend toward a reduction in apoptosis, but this was not statistically significant. We conclude that H. pylori infection is accompanied by the formation of endogenous reactive nitrogen intermediates, which may contribute to DNA damage and apoptosis. In addition to antimicrobial therapy, dietary supplementation with beta-carotene and ascorbic acid may prevent the formation of these potential carcinogens.

Minibody: A Novel Engineered Anti-Carcinoembryonic Antigen Antibody Fragment (Single-Chain Fv-CH3) Which Exhibits Rapid, High-Level Targeting of Xenografts

Abstract: A novel engineered antibody fragment (VL-VH-CH3, or "minibody") with bivalent binding to carcinoembryonic antigen (CEA) was produced by genetic fusion of a T84.66 (anti-CEA) single-chain antibody (scFv) to the human IgG1 CH3 domain. Two designs for the connecting peptide were evaluated. In the T84.66/212 LD minibody, a two-amino acid linker (generated by fusion of restriction sites) was used to join VH and CH3. In the T84.66/212 Flex minibody, the human IgG1 hinge plus an additional 10 residues were used as the connecting peptide. Size exclusion chromatography of purified minibodies demonstrated that both proteins had assembled into Mr80,000 dimers as expected. Furthermore, analysis by SDS-PAGE under nonreducing conditions was consistent with disulfide bond formation in the hinge of the T84.66 Flex minibody. Purified minibodies retained high affinity for CEA (KA, 2 x 10(9) M(-1)) and demonstrated bivalent binding to antigen. Tumor targeting properties were evaluated in vivo using athymic mice bearing LS174T human colon carcinoma xenografts. 123I-labeled T84.66 minibodies demonstrated rapid, high tumor uptake, reaching 17% injected dose/gram (%ID/g) for the LD minibody and 33%ID/g for the Flex minibody at 6 h following injection. Radioiodinated minibody also cleared rapidly from the circulation, yielding high tumor:blood uptake ratios: 44.5 at 24 h for the LD minibody and 64.9 at 48 h for the Flex minibody. Rapid localization by the T84.66/212 Flex minibody allowed imaging of xenografts at 4 and 19 h after administration.

Altered Expression of hMSH2 and hMLH1 in Tumors with Microsatellite Instability and Genetic Alterations in Mismatch Repair Genes

Abstract: To date, at least four genes involved in DNA mismatch repair (MMR) have been demonstrated to be altered in the germline of patients with hereditary nonpolyposis colon cancer: hMSH2, hMLH1, hPMS1, and hPMS2. Additionally, loss of MMR function has been demonstrated to lead to the phenomenon of microsatellite instability (MIN) in tumors from these patients. In this study, we have examined the protein expression pattern of hMSH2 and hMLH1 by immunohistochemistry in paraffin-embedded tumors from 7 patients with MIN+ sporadic cancer, 13 patients with familial colorectal cancer, and 12 patients meeting the strict Amsterdam criteria for hereditary nonpolyposis colon cancer. The relationship between the expression of these two gene products, the presence of germline or somatic mutations, and the presence of tumor MIN was examined. Nineteen of the 28 tumors studied demonstrated MIN, whereas mutations in hMLH1 and hMSH2 were detected in 6 and 2 patients, respectively. Of the eight MIN+/mutation+ cases, the absence of protein expression was observed for the corresponding gene product in all but one case (missense mutation in hMLH1). However, seven MIN+/mutation- cases also showed no expression of either hMLH1 (n = 5), hMSH2 (n = 1), or both (n = 1), whereas four MIN+/mutation- cases demonstrated normal expression for both. None of the MIN-/mutation- cases (n = 9) demonstrated an altered expression pattern for either protein. These data suggest that examination of protein expression by immunohistochemistry may be a rapid method for prescreening tumors for mutations in the MMR genes.