Showing papers in "Cancer Research in 1993"

Specific Proteolytic Cleavage of Poly(ADP-ribose) Polymerase: An Early Marker of Chemotherapy-induced Apoptosis

Abstract: Apoptosis is a morphologically and biochemically distinct form of cell death that occurs under a variety of physiological and pathological conditions. In the present study, the proteolytic cleavage of poly(ADP-ribose) polymerase (pADPRp) during the course of chemotherapy-induced apoptosis was examined. Treatment of HL-60 human leukemia cells with the topoisomerase II-directed anticancer agent etoposide resulted in morphological changes characteristic of apoptosis. Endonucleolytic degradation of DNA to generate nucleosomal fragments occurred simultaneously. Western blotting with epitope-specific monoclonal and polyclonal antibodies revealed that these characteristic apoptotic changes were accompanied by early, quantitative cleavage of the M(r) 116,000 pADPRp polypeptide to an M(r) approximately 25,000 fragment containing the amino-terminal DNA-binding domain of pADPRp and an M(r) approximately 85,000 fragment containing the automodification and catalytic domains. Activity blotting revealed that the M(r) approximately 85,000 fragment retained basal pADPRp activity but was not activated by exogenous nicked DNA. Similar cleavage of pADPRp was observed after exposure of HL-60 cells to a variety of chemotherapeutic agents including cis-diaminedichloroplatinum(II), colcemid, 1-beta-D-arabinofuranosylcytosine, and methotrexate; to gamma-irradiation; or to the protein synthesis inhibitors puromycin or cycloheximide. Similar changes were observed in MDA-MB-468 human breast cancer cells treated with trifluorothymidine or 5-fluoro-2'-deoxyuridine and in gamma-irradiated or glucocorticoid-treated rat thymocytes undergoing apoptosis. Treatment with several compounds (tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, N-ethylmaleimide, iodoacetamide) prevented both the proteolytic cleavage of pADPRp and the internucleosomal fragmentation of DNA. The results suggest that proteolytic cleavage of pADPRp, in addition to being an early marker of chemotherapy-induced apoptosis, might reflect more widespread proteolysis that is a critical biochemical event early during the process of physiological cell death.

The "bystander effect": tumor regression when a fraction of the tumor mass is genetically modified.

Abstract: Tumor cells expressing the herpes simplex virus thymidine kinase (HSV-TK) gene are sensitive to the drug ganciclovir (GCV). We demonstrate here that HSV-TK-positive cells exposed to GCV were lethal to HSV-TK-negative cells as a result of a "bystander effect." HSV-TK-negative cells were killed in vitro when the population of cultured cells contained only 10% HSV-TK-positive cells. The mechanism of this "bystander effect" on HSV-TK-negative cells appeared to be related to the process of apoptotic cell death when HSV-TK-positive cells were exposed to GCV. Flow cytometric and electron microscopic analyses suggested that apoptotic vesicles generated from the dying gene-modified cells were phagocytized by nearby, unmodified tumor cells. Prevention of apoptotic vesicle transfer prevented the bystander effect. The toxic effect of HSV-TK-positive cells on HSV-TK-negative cells was reproduced in an in vivo model. A mixed population of tumor cells consisting of HSV-TK-positive and HSV-TK-negative cells was inoculated s.c. into mice. Regression of the tumor mass occurred when the inoculum consisted of as few as 10% HSV-TK-expressing tumor cells. The bystander effect was also demonstrated in i.p. tumor studies. Initial experiments demonstrated that prolonged survival (> 70 days) occurred when a mixture containing 50% HSV-TK-positive and 50% HSV-TK-negative cells was injected i.p. followed by GCV treatment. Further, survival was prolonged for mice with a preexisting HSV-TK-negative i.p. tumor burden by injecting HSV-TK-positive cells and GCV. These results suggest that genetic modification of tumor cells may be useful for developing cancer therapies.

Investigation of the Subcellular Distribution of the bcl-2 Oncoprotein: Residence in the Nuclear Envelope, Endoplasmic Reticulum, and Outer Mitochondrial Membranes

Abstract: A multidisciplinary approach was taken to investigate the intracellular locations of the 26-kDa integral membrane protein encoded by the bcl-2 gene. Subcellular fractionation analysis of a t(14;18)-containing lymphoma cell line revealed the presence of Bcl-2 protein in nuclear, heavy-membrane, and light-membrane fractions but not in cytosol. Sedimentation of heavy-membrane fractions in Nycodenz and Percoll continuous gradients demonstrated comigration of p26-Bcl-2 with mitochondrial but not other organelle-associated proteins. Fractionation of light-membrane fractions using discontinuous sucrose-gradients revealed association of Bcl-2 protein primarily with lighter-density microsomes (smooth endoplasmic reticulum) as opposed to heavy-density microsomes (rough endoplasmic reticulum). Immune microscopy studies using laser-scanning microscopy, pre- and postembedding electron microscopic methods, and six different anti-Bcl-2 antibodies demonstrated Bcl-2 immunoreactivity in the nuclear envelope and outer mitochondrial membrane in a patchy distribution. Furthermore, anti-Bcl-2 antibody immunoreactivity generally appeared to directly overlie the nuclear envelope in high magnification electron microscopic studies, reminiscent of nuclear pore complexes. Addition of in vitro translated p26-Bcl-2 to isolated translocation-competent mitochondria revealed transmembrane domain-dependent association of Bcl-2 protein with mitochondria but provided no evidence for import into a protease-resistant compartment, consistent with immunomicroscopic localization to the outer mitochondrial membrane. Taken together, the findings demonstrate that p26-Bcl-2 resides primarily in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membrane in a nonuniform distribution suggestive of participation in protein complexes perhaps involved in some aspect of transport.

Detection of DNA Strand Breaks in Individual Apoptotic Cells by the in Situ Terminal Deoxynucleotidyl Transferase and Nick Translation Assays

Abstract: DNA strand breaks which occur in HL-60 cells as a result of activation of endonuclease during apoptosis induced by cell treatment with the DNA topoisomerase I inhibitor camptothecin and topoisomerase II inhibitors teniposide, 4′-(9-acridinylamino)-3-methanesulfon-m-anisidide, and fostriecin were labeled in situ, in individual fixed and permeabilized cells, with biotinylated dUTP (detected by fluoresceinated avidin), using the terminal deoxynucleotidyl transferase or nick translation assays During the early stage of apoptosis, prior to nuclear fragmentation, the breaks were predominantly localized at the nuclear periphery, close to the nuclear envelope In more advanced stages, all cellular DNA, then localized within the cell as dense, homogeneous granules of a variety of sizes, was strongly labeled, indicating extensive and more uniform distribution of breaks throughout genomic DNA Bivariate analysis of the incorporated biotinylated dUTP and cellular DNA content by flow cytometry made it possible to estimate the kinetics of the labeling reaction and relate DNA breaks to cell position in the cycle The kinetics of biotinylated dUTP incorporation was faster, and the distinction of cells with DNA breaks was more pronounced, using the terminal transferase rather than the nick translation assay Camptothecin, teniposide, and 4′-(9-acridinylamino)-3-methanesulfon-m-anisidide induced DNA breaks preferentially in S-phase cells, having little effect on cells in the G1 phase of the cycle In contrast, fostriecin affected cells indiscriminately, in all phases of the cell cycle The method of detection of DNA strand breaks (3′-hydroxyl termini) individual cells offers several advantages and can be applied to clinical material (tumor biopsies) to study the induction of apoptosis in tumors during treatment, as a possible prognostic marker The protein-associated DNA breaks in the “cleavable” DNA-topoisoerase complexes, which are the primary lesions induced by the inhibitors and precede apoptosis, were not detectable by the present methods

Aspirin Use and Risk of Fatal Cancer

Abstract: Aspirin and other nonsteroidal antiinflammatory drugs inhibit prostaglandin synthesis and tumor growth in many experimental systems, but it is unclear which of these tumor models are relevant to humans. We have reported reduced risk of fatal colon cancer among persons who used aspirin in a large prospective study. This analysis examines other fatal cancers in relation to aspirin among 635,031 adults in that study who provided information in 1982 on the frequency and duration of their aspirin use and did not report cancer. Death rates were measured through 1988. Death rates decreased with more frequent aspirin use for cancers of the esophagus, stomach, colon, and rectum but not generally for other cancers. For each digestive tract cancer, death rates were approximately 40% lower among persons who used aspirin 16 times/month or more for at least 1 year compared to those who used no aspirin. The trend of decreasing risk with more frequent aspirin use was strongest among persons who had used aspirin for 10 years or more; it remained statistically significant, except for esophageal cancer, in multivariate analyses that adjusted for other known risk factors. Biases such as early detection or aspirin avoidance among cases do not appear to explain the results. Our data suggest that regular, prolonged use of aspirin may reduce the risk of fatal cancer of the esophagus, stomach, colon, and rectum. Future epidemiological and basic research should examine all digestive tract cancers in considering the chemopreventive or therapeutic potential of nonsteroidal antiinflammatory drugs.

Elevated Levels of Transforming Growth Factor α and Epidermal Growth Factor Receptor Messenger RNA Are Early Markers of Carcinogenesis in Head and Neck Cancer

Abstract: The squamous mucosa of patients who develop head and neck cancer is “condemned” or predisposed to disregulated growth as reflected by the high incidence of synchronous and metachronous primary tumors. We hypothesized that transformed and nontransformed mucosa from head and neck cancer patients would produce increased levels of transforming growth factor α (TGF-α) and its cell surface receptor, the epidermal growth factor receptor (EGFR), thereby contributing to this predisposition. Using molecular biological techniques, we examined the incidence and mechanism of TGF-α and EGFR overproduction in tumors and histologically normal mucosa excised from patients with squamous cell carcinoma of the head and neck (SCCHN) to test this hypothetical mechanism of field cancerization. Northern blot hybridization was used to evaluate the frequency of increased TGF-α and EGFR mRNA production in tissue excised from 24 patients with SCCHN and 10 cell lines compared with 7 control patients without cancer or a history of alcohol and tobacco use. Southern blot hybridization was used to examine for gene amplification. In patients with SCCHN, TGF-α mRNA was elevated by a mean of 5-fold in 95% of histologically “normal” mucosa samples ( P = 0.001) and by a mean of 5-fold in 87.5% of tumors ( P = 0.0001) while EGFR mRNA was elevated by a mean of 29-fold in 91% of histologically normal mucosa specimens ( P = 0.0005) and by a mean of 69-fold in 92% of tumors ( P = 0.0005), compared with mRNA levels in control normal mucosa. In 10 SCCHN cell lines, TGF-α mRNA was increased by a mean of 16-fold and EGFR mRNA levels were increased by a mean of 77-fold. Increased production of TGF-α and EGFR mRNA in the histologically normal mucosa of patients at risk for a primary or secondary head and neck cancer may serve both as a marker for malignant transformation and as a target for preventive therapies.

Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history.

Abstract: Recent reports have suggested that one or more genes may cause replication errors (RER) during colorectal tumorigenesis. Additional alleles are seen in the tumors when analyzing random microsatellite loci. We have studied seven dinucleotide repeat loci, located on seven different chromosomes, by use of polymerase chain reaction amplification and denaturing polyacrylamide gel electrophoresis. We found that 16.5% (40 of 243) colorectal cancers showed RER at one or several loci (RER+). This includes 31% (4 of 13) among cases with a strong positive family history according to previously published criteria and 17% (35 of 207) among cases with no history of familial cancer. Interestingly, no significant association was found between RER+ tumors and a general familial clustering of cancer. Microsatellite instability was significantly associated with DNA diploid status of the tumor ( P P P P = 0.05). We further analyzed 84 breast cancers and 86 male germ cell cancers using the same seven markers. None of the tumors were RER+, indicating that this phenomenon may be specific to certain types of tumors.

Expression of Vascular Permeability Factor (Vascular Endothelial Growth Factor) and Its Receptors in Adenocarcinomas of the Gastrointestinal Tract

Abstract: Vascular permeability factor (VPF) is one of the most potent known inducers of microvascular hyperpermeability; in addition, it is a selective endothelial cell growth factor, hence its alternate name, vascular endothelial growth factor. VPF exerts its actions on the microvasculature by interacting with specific endothelial cell receptors. VPF is expressed by many transplantable animal tumors, by tumor cell lines in culture, and by certain normal cells in situ. The purpose of the present investigation was to determine whether and with what consistency VPF and its endothelial cell receptors are expressed in primary autochthonous human tumor of gastrointestinal tract origin, as determined by in situ hybridization and immunohistochemistry. Twenty-one primary adenocarcinomas (17 colon, 2 stomach, 1 small bowel, and 1 pancreas) were studied. The malignant epithelial cells expressed VPF mRNA strongly, in contrast to normal epithelium, hyperplastic polyps, and adenomas, which expressed little or no VPF mRNA. VPF expression was further increased in tumor cells immediately adjacent to zones of tumor necrosis; in such areas, occasional stromal cells also expressed VPF mRNA. In the ten colon carcinomas studied, tumor cells stained for VPF protein by immunohistochemistry. The endothelial cells of nearby stromal blood vessels also stained for VPF by immunohistochemistry and in addition expressed mRNAs encoding the VPF receptors flt-1 and kdr as determined by in situ hybridization. Endothelial cells away from the tumor did not stain for VPF and no definite mRNA expression for flt-1 or kdr was detected by in situ hybridization. The ganglion cells of the myenteric plexus of normal bowel expressed VPF mRNA and protein. These data indicate that primary autochthonous human tumors of gastrointestinal origin regularly express both VPF mRNA and VPF protein and that adjacent stromal vessels express mRNAs for both known VPF receptors. VPF is likely to contribute to tumor growth by promoting angiogenesis and stroma formation, both directly, through its action as an endothelial cell growth factor, and indirectly, by increasing vascular permeability, thereby leading to plasma protein extravasation, fibrin deposition, and the eventual replacement of the resulting matrix with vascularized stroma.

Expression of E-cadherin cell adhesion molecules in human breast cancer tissues and its relationship to metastasis

Abstract: E-cadherin (E-cad) is a subclass of the cadherin family that plays a major role in maintenance of intercellular junctions in epithelial tissues In order to explore the correlation between the expression of E-cad and cancer invasion and metastasis in vivo, we performed an immunohistochemical examination for E-cad expression in 120 patients with breast cancer using our specific anti-E-cad monoclonal antibody In noncancerous epithelial cells, E-cad was strongly expressed on cell-cell boundaries, whereas various staining patterns were observed in tumors Of these 120 tumors, 56 (47%) showed Pr type expression of E-cad, and 64 (53%) showed Rd type or negative expression We found significant correlations between E-cad expression and clinicopathological features The frequency of Rd type was significantly higher in invasive ductal carcinomas (58%, 56 of 97) and poorly differentiated carcinomas (84%, 21 of 25) than in noninvasive and well-differentiated carcinomas Furthermore, a high frequency of Rd type was detected in the following advanced tumors: T3,4 tumors, 71% (22 of 31); tumors with extensive lymph node metastasis, 74% (29 of 39); and tumors with distant metastasis, 86% (19 of 22) These values were significantly higher compared with their counterparts The expression of epidermal growth factor receptor tended to be positive in E-cad-positive tumors However, no significant relationship was seen among E-cad expression, menopausal status, hormone receptor status, and DNA ploidy pattern These results suggest that the reduction of E-cad expression may play an important role in invasion and metastasis of human breast cancer

Heterogeneity of Mucin Gene Expression in Normal and Neoplastic Tissues

Abstract: To determine the relative expression of distinct mucin genes in normal and neoplastic tissue, antibodies and cDNA probes that recognize the core tandem repeat sequences of membrane-bound (MUC1) and secreted (MUC2 and MUC3) mucins were used for immunohistochemical and RNA Northern and slot-blot analysis. MUC1 mRNA was detected in all epithelial tissues tested. MUC1 core peptide, recognized by monoclonal antibodies 139H2 and DF3, was highly expressed on apical membranes of bronchus, breast, salivary gland, pancreas, prostate, and uterus, and was sparsely expressed in gastric surface cells, gallbladder, small intestine, and colonic epithelium. In contrast, MUC2 and MUC3 gene expression was primarily restricted to the intestinal tract. MUC2 mRNA was highly expressed in normal jejunum, ileum, and colon, compared with very low levels in normal bronchus and gallbladder. MUC3 mRNA was highly expressed in normal jejunum, ileum, colon, and gallbladder. Immunohistochemical studies using antibodies against synthetic MUC2 (anti-MRP) and MUC3 (anti-M3P) peptides indicate that MUC2- and MUC3-producing cells in the gastrointestinal tract are distinct. Goblet cells of the small intestine and colon reacted strongly with anti-MRP, whereas M3P reactivity was restricted to columnar cells of small intestinal villi, surface colonic epithelium, and gallbladder. Mucin protein epitopes and mRNA levels were frequently altered in adenocarcinomas compared to corresponding normal tissues. Alterations included increased expression, aberrant expression, and, less frequently, loss of expression. Increased MUC1 immunoreactivity was observed in most adenocarcinomas of the breast, lung, stomach, pancreas, prostate, and ovary. In addition, with the exception of prostate cancer, focal aberrant expression of MUC2 and MUC3 epitopes was frequently observed. Increased MUC1, MUC2, and MUC3 epitopes were present in colon adenocarcinomas of all histological subtypes, with the greatest increase of MUC2 epitopes observed in colloid (mucinous) colon cancers. MUC2 or MUC3 mRNA levels were increased in colloid colon cancer compared with normal colon, however in well- and moderately well-differentiated colon cancers MUC1, 2 and 3 mRNA levels were decreased. Compared with corresponding normal tissue, MUC1 mRNA levels were increased in breast cancer and well-differentiated lung cancers, and MUC3 mRNA was increased in gastric adenocarcinomas. Normal stomach lacked both MUC2 and MUC3 immunoreactivity and mRNA, however, MUC2 and MUC3 proteins and mRNA were highly expressed in gastric intestinal metaplasia. In conclusion, mucin genes are independently regulated and their expression is organ- and cell type-specific. Furthermore, neoplastic transformation is associated with dys-regulated expression of both membrane-bound and secreted mucin core protein epitopes and may be due to altered mucin mRNA levels and/or altered mucin glycosylation.

The Incidence of p53 Mutations Increases with Progression of Head and Neck Cancer

Abstract: To establish a genetic model of the progression of head and neck squamous carcinoma we have defined the incidence and timing of p53 mutations in this type of cancer. We sequenced the conserved regions of the p53 gene in 102 head and neck squamous carcinoma lesions. These included 65 primary invasive carcinomas and 37 noninvasive archival specimens consisting of 13 severe dysplasias and 24 carcinoma in situ lesions. The incidence of p53 mutations in noninvasive lesions was 19% (7/37) and increased to 43% (28/65) in invasive carcinomas. These data suggest that p53 mutations can precede invasion in primary head and neck cancer. Furthermore, the spectrum of codon hotspots is similar to that seen in squamous carcinoma of the lung and 64% of mutations are at G nucleotides, implicating carcinogens from tobacco smoke in the etiology of head and neck squamous carcinoma.

Microsatellite instability is associated with tumors that characterize the hereditary non-polyposis colorectal carcinoma syndrome.

Abstract: Microsatellite instability implying multiple replication errors (RER+ phenotype) characterizes a proportion of colorectal carcinomas, particularly those from patients with the hereditary non-polyposis colorectal carcinoma syndrome. We studied the incidence of microsatellite instability in more than 500 sporadic tumors representing 6 different types of cancer. Apart from colorectal carcinoma [see the paper by Lothe et al. (Cancer Res., 53:5849-5852, 1993)] the RER+ phenotype was found in 18% (6 of 33) of gastric carcinomas and 22% (4 of 18) of endometrial carcinomas. In contrast, no evidence of this abnormality was detected in cancers of the lung (N = 85), breast (N = 84), and testis (N = 86). Importantly, the first three cancers, as opposed to the latter three, are characteristic of the hereditary non-polyposis colorectal carcinoma syndrome. These findings suggest that the cancers belonging to the hereditary non-polyposis colorectal carcinoma tumor spectrum may have essential pathogenetic steps in common, including a tendency to multiple replication errors.

Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations.

Abstract: The MDM2 (murine double minute 2) gene has recently been shown to code for a cellular protein that can complex the p53 tumor suppressor gene product and inhibit its function. We studied a series of 157 primary brain tumors and report here that the MDM2 gene is amplified and overexpressed in 8-10% of glioblastomas and anaplastic astrocytomas. Thus, MDM2 represents the second most frequently amplified gene after the epidermal growth factor receptor gene in these tumor types. Sequencing of the p53 transcripts in the cases with MDM2 amplification revealed no mutations and restriction fragment length polymorphism analysis showed, with one exception, no losses of alleles on chromosome 17. Our results indicate that amplification and overexpression of MDM2 may be an alternative molecular mechanism by which a subset of human malignant gliomas escapes from p53-regulated growth control.

Expression of CD44 Variant Proteins in Human Colorectal Cancer Is Related to Tumor Progression

Abstract: Specific CD44 variant glycoproteins are overexpressed at particular stages of colorectal tumor progression. Some variants of the CD44 glycoprotein without exon v6 sequences appear at the earliest stage of tumorigenesis, i.e., in early adenomas. Expression of variants containing exon v6 sequences is largely restricted to the advanced stages of tumor development and in addition is more prevalent and intense in metastatic (Dukes C/D) than in nonmetastatic (Dukes A/B) carcinomas. The observation that CD44 variants containing a protein domain of CD44 that confers full metastatic potential to rat carcinoma and sarcoma cell lines is increasingly expressed during colorectal tumor progression indicates that this domain may have an important role in tumor progression and metastasis in humans. Information on v6 expression, which can be obtained by routine immunohistochemistry, may prove of important prognostic value, particularly in carcinomas (Dukes A and B) that have not yet given rise to detectable metastases.

Molecular Cloning of a Complementary DNA Encoding a Prostate-specific Membrane Antigen

Abstract: Recently, a novel M(r) 100,000 prostate-specific membrane glycoprotein (PSM) has been detected by the prostate-specific monoclonal antibody 7E11-C5, raised against the human prostatic carcinoma cell line LNCaP. The PSM antigen is expressed exclusively by normal and neoplastic prostate cells and metastases. We now report the molecular cloning of a full-length 2.65-kilobase complementary DNA encoding the PSM antigen from a human LNCaP complementary DNA library by polymerase chain reaction using degenerate oligonucleotide primers. Analysis of the complementary DNA sequence has revealed that a portion of the coding region, from nucleotide 1250 to 1700, has 54% homology to the human transferrin receptor mRNA. The deduced polypeptide has a putative transmembrane domain enabling the delineation of intra- and extracellular portions of this antigen. In contrast to prostate-specific antigen and prostatic acid phosphatase which are secreted proteins, PSM as an integral membrane protein may prove to be effective as a target for imaging and cytotoxic targeting modalities.

Contribution of Carbohydrate Antigens Sialyl Lewis A and Sialyl Lewis X to Adhesion of Human Cancer Cells to Vascular Endothelium

Abstract: The carbohydrate antigen, sialyl Lex, is known to be a ligand for the cell adhesion molecule called ELAM-1 (E-selectin, endothelial cell leukocyte adhesion molecule-1), which is present on cytokine-activated human endothelial cells. Recently, we reported that another carbohydrate antigen, sialyl Lea, can also serve as a ligand for ELAM-1 (A. Takada, K. Ohmori, N. Takahashi, K. Tsuyuoka, K. Yago, K. Zenita, A. Hasegawa, and R. Kannagi, Biochem. Biophys. Res. Commun., 179: 713-719, 1991). Both sialyl Lex and sialyl Lea are expressed in many human malignant cells. In order to assess the contribution of these carbohydrate antigens to the adhesion of human malignant cells to vascular endothelium, we selected a panel of 12 cultured human epithelial cancer cell lines and a panel of 12 human leukemia cell lines which express sialyl Lex and/or sialyl Lea antigens. All 12 epithelial cancer cell lines exhibited a clearly ELAM-1-dependent adhesion to cytokine-activated human umbilical vein endothelial cells, while only 3 of the 12 leukemia cell lines exhibited significant participation of ELAM-1 in the adhesion. With regard to epithelial cancer cells, the adhesion of 6 cancer cell lines, mostly of colon and pancreas origin, was dependent almost exclusively on sialyl Lea. A significant contribution of the sialyl Lex antigen was noted in the adhesion of the other 6 cell lines, including cancers of lung and liver origin. These results imply that the sialyl Lea/ELAM-1 adhesion system, as well as the sialyl Lex/ELAM-1 adhesion system, plays an important role in the adhesion of human cancer cells to human umbilical vein endothelial cells. With regard to leukemia cells, on the other hand, adhesion of the 3 leukemia cell lines that showed ELAM-1-dependent adhesion was mediated by the sialyl Lex antigen, and none of these leukemia cell lines expressed sialyl Lea or exhibited sialyl Lea-dependent adhesion.

p53 Mutation and MDM2 Amplification in Human Soft Tissue Sarcomas

Abstract: The p53 and MDM2 genes were analyzed in 24 human soft tissue sarcomas (11 malignant fibrous histiocytomas and 13 liposarcomas). Alterations of p53, consisting of point mutations, deletions, or overexpression, were detected in one-third (8 of 24) of the sarcomas. MDM2 gene amplification was detected in another 8 tumors, but no tumor contained an alteration of both genes. Monoclonal antibodies reactive with the human MDM2 gene product were developed, and immunohistochemical analysis revealed nuclear localization and overexpression of MDM2 in those tumors with amplified MDM2 genes. These data support the hypothesis that p53 and MDM2 genetic alterations are alternative mechanisms for inactivating the same regulatory pathway for suppressing cell growth.

Genetic instability of microsatellites in endometrial carcinoma.

Abstract: Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by a familial predisposition to colorectal carcinoma and extracolonic cancers of the gastrointestinal, urological, and female reproductive tracts, notably the endometrium. A genetic locus for HPNCC was recently determined by linkage analysis to exist on chromosome 2p; both sporadic and HNPCC-associated colorectal carcinomas exhibit a "replication error" phenotype, characterized by instability of dinucleotide repeat sequences throughout the genome. Here, we address the hypothesis that the replication error phenotype would be evident in some fraction of sporadic endometrial carcinomas or in those associated with HNPCC. Microsatellite instability was observed in 17% of sporadic endometrial carcinomas and in 75% of those tumors associated with HNPCC. These data indicate that the HNPCC gene is also involved in heritable and somatic forms of endometrial carcinoma.

Differential Activation of Cyclophosphamide and Ifosphamide by Cytochromes P-450 2B and 3A in Human Liver Microsomes

Abstract: The present study identifies the specific human cytochrome P-450 (CYP) enzymes involved in hydroxylation leading to activation of the anticancer drug cyclophosphamide and its isomeric analogue, ifosphamide. Substantial interindividual variation (4-9-fold) was observed in the hydroxylation of these oxazaphosphorines by a panel of 12 human liver microsomes, and a significant correlation was obtained between these 2 activities ( r = 0.85, P K m and high K m P-450 4-hydroxylases. To ascertain whether one or more human P-450 enzymes are catalytically competent in activating these oxazaphosphorines, microsomal fractions prepared from a panel of human B-lymphoblastoid cell lines stably transformed with individual P-450 complementary DNAs were assayed in vitro for oxazaphosphorine activation. Expressed CYP2A6, -2B6, -2C8, -2C9, and -3A4 were catalytically competent in hydroxylating cyclophosphamide and ifosphamide. Whereas CYP2C8 and CYP2C9 have the characteristics of low K m oxazaphosphorine 4-hydroxylases, CYP2A6, -2B6, and -3A4 are high K m forms. In contrast, CYP1A1, -1A2, -2D6, and -2E1 did not produce detectable activities. Furthermore, growth of cultured CYP2A6- and CYP2B6-expressing B-lymphoblastoid cells, but not of CYP-negative control cells, was inhibited by cyclophosphamide and ifosphamide as a consequence of prodrug activation to cytotoxic metabolites. Experiments with P-450 form-selective chemical inhibitors and inhibitory anti-P-450 antibodies were then performed to determine the contributions of individual P-450s to the activation of these drugs in human liver microsomes. Orphenadrine (a CYP2B6 inhibitor) and anti-CYP2B IgG inhibited microsomal cyclophosphamide hydroxylation to a greater extent than ifosphamide hydroxylation, consistent with the 8-fold higher activity of complementary DNA-expressed CYP2B6 with cyclophosphamide. In contrast, troleandomycin, a selective inhibitor of CYP3A3 and -3A4, and anti-CYP3A IgG substantially inhibited microsomal ifosphamide hydroxylation but had little or no effect on microsomal cyclophosphamide hydroxylation. By contrast, the CYP2D6-selective inhibitor quinidine did not affect either microsomal activity, while anti-CYP2A antibodies had only a modest inhibitory effect. Overall, the present study establishes that liver microsomal CYP2B and CYP3A preferentially catalyze cyclophosphamide and ifosphamide 4-hydroxylation, respectively, suggesting that liver P-450-inducing agents targeted at these enzymes might be used in cancer patients to enhance drug activation and therapeutic efficacy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Up-Regulation of Vascular Endothelial Growth Factor and Its Cognate Receptors in a Rat Glioma Model of Tumor Angiogenesis

Abstract: We have recently shown that vascular endothelial growth factor (VEGF) is produced by human malignant glioma cells and acts on tumor endothelial cells, which express VEGF receptors, suggesting that VEGF is a regulator of tumor angiogenesis. To investigate the feasibility of antiangiogenic brain tumor therapy, we developed an intracerebral (i.c.) rat glioma model. We used two transplantable rat glioma cells lines, C6 and GS-9L, to analyze VEGF regulation in vitro and expression of VEGF and its high affinity tyrosine kinase receptors, flt-1 and flk-1, in vivo. Glioma cells were transplanted i.c. or s.c. into syngeneic rats. C6 gliomas exhibit morphological characteristics of human glioblastoma multiforme such as necroses with palisading cells. Immunocytochemistry with von Willebrand factor showed that C6 gliomas are highly vascularized and therefore show another prominent feature of human glioblastoma. GS-9L gliosarcomas were less vascularized. In situ hybridization showed that VEGF is expressed in vivo in rat glioma cells which reside along necrotic areas and therefore closely mimicks the expression pattern of VEGF observed in human glioblastoma. flt-1 and flk-1 are specifically expressed in endothelial cells in the tumor and at the border between tumor and normal brain but are absent from endothelial cells in the normal brain proper. The action of VEGF may therefore be restricted to tumor endothelium. Upregulation of VEGF, but not acid fibroblast growth factor, basic fibroblast growth factor, and platelet-derived growth factor B messenger RNA was observed in hypoxic C6 and GS-9L cells in vitro. These observations are consistent with a role for VEGF in tumor- and hypoxia-induced angiogenesis. Since the expression pattern of VEGF and its receptors in rat glioma appears to be indistinguishable from human glioblastoma multiforme, this model provides an excellent tool to study anti-angiogenic therapy.

In Vitro and in Vivo Reversal of Multidrug Resistance by GF120918, an Acridonecarboxamide Derivative

Abstract: N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]- phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) has been selected from a chemical program aimed at identifying an optimized inhibitor of multidrug resistance (MDR). The potency of GF120918 is assessed by dose-dependent sensitization of CHRC5, OV1/DXR and MCF7/ADR cells to the cytotoxicity of doxorubicin and vincristine respectively: GF120918 fully reverses multidrug resistance at 0.05 to 0.1 microM and is half maximally active at 0.02 microM. The spectrum of drugs sensitized by GF120918 coincides with those having the classical MDR phenotype. In CHRC5 cells, 0.01-0.1 microM GF120918 enhances the uptake of [3H]daunorubicin and blocks the efflux from preloaded cells. It is also shown that GF120918 is still active several hours after being taken away from the culture medium showing that it is not, like verapamil, effluxed rapidly by P-glycoprotein. GF120918 effectively competes with [3H]azidopine for binding P-glycoprotein, pointing to this transport membrane protein as its likely site of action. After i.v. administration to mice, GF120918 penetrates thoroughly various organs that have a tissue level/blood level ratio above 10. It is eliminated from organs and blood with a half-time of approximately 2.7 h. It is well absorbed after p.o. administration. In mice implanted i.p. with the MDR P388/Dox tumor, a single i.v. or p.o. dose of GF120918 restores sensitivity of the tumor to a single i.p. dose (5 mg/kg) of doxorubicin administered 1 h later. A statistically significant effect is observed at 1 mg/kg GF120918 i.v. and maximal effect is reached at 5 mg/kg. Similarly, whereas neither drug alone is effective, GF120918 (10 mg/kg i.p.) associated with doxorubicin (5 mg/kg i.p.) inhibits the growth of the moderately MDR C26 tumor implanted s.c. as assessed by tumor size at day 19. GF120918 does not modify significantly the distribution or the elimination of doxorubicin in mice ruling out the possibility that the antitumor effects seen might be explained by pharmacokinetic interactions.

Functional imaging of multidrug-resistant P-glycoprotein with an organotechnetium complex.

Abstract: The multidrug-resistant P-glycoprotein (Pgp), a M r 170,000 plasma membrane protein encoded by the mammalian multidrug resistance gene ( MDRI ), appears to function as an energy-dependent efflux pump. Many of the drugs that interact with Pgp are lipophilic and cationic at physiological pH. We tested the hypothesis that the synthetic γ-emitting organotechnetium complex, hexakis(2-methoxyisobutylisonitrile)technetium(I) ([ 99m Tc]SESTAMIBI), a lipophilic cationic radiopharmaceutical, could be a suitable Pgp transport substrate capable of functional imaging of the MDR phenotype. The cellular pharmacological profile of [ 99m Tc]SESTAMIBI transport was examined in Chinese hamster V79 lung fibroblasts and the 77A and LZ derivative cell lines which express modestly low, intermediate, and very high levels of Pgp, respectively. Steady-state contents of [ 99m Tc]SESTAMIBI in V79, 77A, and LZ cells were 10.0 ± 0.5 (SEM) ( n = 9), 3.6 ± 0.5 ( n = 8), and 0.4 ± 0.02 ( n = 9) fmol·(mg protein) -1 (nm o ) -1 , respectively, consistent with enhanced extrusion of the imaging agent by Pgp-enriched cells. Maximal doses (>100 µm) of the multidrug-resistant reversal agents verapamil and cyclosporin A enhanced [ 99m Tc]SESTAMIBI accumulation in V79, 77A, and LZ cells by approximately 10-, 25-, and 200-fold, respectively. The median effective concentration values for tracer accumulation in the presence of verapamil in V79, 77A, and LZ cells were 4, 100, and 200 µm, and those for cyclosporin A were 0.9, 3, and >25 µm, respectively. Pgp-mediated [ 99m Tc]SESTAMIBI transport occurred against its electrochemical gradient and was found to be ATP dependent displaying an apparent K m of 50 µm. Carrier-added [ 99 Tc]SESTAMIBI was 11- to 13-fold less toxic in multidrug-resistant cells, and inhibited photolabeling of Pgp by [ 125 I]iodoaryl azidoprazosin in a concentration-dependent manner; half-maximal displacement was observed at approximately 100- to 1000-fold molar excess [ 99 Tc]SESTAMIBI. Exploiting the favorable γ emission properties of 99m Tc, functional expression of Pgp was successfully imaged in human tumor xenographs in nude mice with pharmacologically inert tracer quantities of [ 99m Tc]SESTAMIBI. Functional imaging with these organotechnetium complexes may provide a novel mechanism to rapidly characterize Pgp expression in human tumors in vivo , target reversal agents in vivo , and ultimately provide a means to direct patients to specific cancer therapies.

In Situ Retroviral-mediated Gene Transfer for the Treatment of Brain Tumors in Rats

Abstract: Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. This suggests that retroviral-mediated gene transfer might permit targeting of gene integration into malignant cells in organs composed mainly of quiescent nonproliferating cells, such as in the brain. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs ("suicide" genes) into proliferating brain tumors may be used to treat this cancer. We investigated the efficacy and dynamics of in vivo transduction of growing brain tumors with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir. Ganciclovir is phosphorylated by thymidine kinase to toxic triphosphates that interfere with DNA synthesis, resulting in the preferential death of the transduced tumor cells. Rats inoculated with 4 x 10(4) 9L gliosarcoma cells into the frontal lobe were treated 7 days later with an intratumoral stereotaxic injection of murine fibroblasts (NIH 3T3 cells) that were producing a retroviral vector containing the herpes simplex-thymidine kinase gene. Controls received vector producer and nonproducer NIH 3T3 cell lines containing the Escherichia coli lacZ (beta-galactosidase) gene as well as nonproducer NIH 3T3 cells containing the thymidine kinase gene. The animals were rested for 7 days to allow time for in situ transduction of the proliferating tumor cells with the herpes-thymidine kinase retroviral vector. The animals were then treated with ganciclovir, 15 mg/kg i.p. twice a day for 14 days. Gliomas receiving an injection of 3-5 x 10(6) thymidine kinase producer cells regressed completely in 23 of 30 rats given ganciclovir therapy, while 25 of 26 control rats developed large tumors. Intratumoral injection of a lower concentration of thymidine kinase vector producer cells (1.8 x 10(6)) resulted in a lower frequency of tumor regression (5 of 13 rats). To estimate the efficiency of in vivo gene transfer, 9L brain tumors were given injections of 5 x 10(6) beta-galactosidase vector producer cells. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranaside staining revealed maximal staining of beta-galactosidase within the tumor 7-14 days after injection of the vector producer cells. In vivo transduction rates in harvested tumors ranged from 10 to 70%. There was no evidence of transduction of the surrounding normal neural tissue. Occasional blood vessel endothelial cells within or adjacent to the tumor were observed to be 5-bromo-4- chloro-3-indolyl-beta-D-galactopyranaside positive.(ABSTRACT TRUNCATED AT 400 WORDS)

Overexpression of a M(r) 110,000 vesicular protein in non-P-glycoprotein-mediated multidrug resistance.

Abstract: A M r110,000 protein (p110) is overexpressed in P-glycoprotein-negative multidrug-resistant tumor cell lines of different histogenetic origins. These cell lines show an ATP-dependent drug accumulation defect, suggesting the presence of drug transporter molecules different from P-glycoprotein. Immunohistochemical staining with a p110-specific monoclonal antibody (LRP-56) showed that, like P-glycoprotein, the molecule has a high expression in normal epithelial cells and tissues chronically exposed to xenobiotics and potentially toxic agents, such as bronchial cells, cells lining the intestines, and kidney tubules. Staining of LRP-56 is primarily cytoplasmic, in a coarsely granular fashion, indicating that it reacts with a molecule closely associated with vesicular/lysosomal structures. Involvement of p110 in the energy-dependent drug transport process present in the cell lines is unknown.

Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung

Abstract: The epidermal growth factor receptor (EGFR) and one of its ligands, transforming growth factor alpha (TGF-alpha), are thought to function as a potential autocrine loop in non-small cell lung cancer (NSCLC). However, the expression pattern of EGFR and the TGF-alpha-related ligands have not been fully characterized in primary NSCLC and adjacent benign lung tissue. For this reason, we comprehensively examined the coexpression and differential expression of EGFR and its ligands, TGF-alpha, epidermal growth factor (EGF), and amphiregulin (AR), by Northern analysis, in paired samples of primary tumors and uninvolved lung. For those RNA species overexpressed in malignant lung, single cell expression patterns were studied by immunohistochemistry. Specimens were obtained from 57 consecutive patients who underwent resection of carefully staged resectable NSCLC and were followed prospectively. Most (112 of 114) tissue samples yielded high-quality RNA. EGFR was expressed in 82 of 88 (93%) tissue samples, while TGF-alpha was expressed in 62 of 72 (86%) samples, and AR was expressed in 64 of 70 (92%) samples. EGF was unexpressed in total cellular RNA in both tumor and uninvolved lung. In a comparison of RNA expression patterns in tumors and uninvolved lung, overexpression of EGFR was found in 45% (22 of 44) of tumors, while overexpression of TGF-alpha was seen in 61% (22 of 36) of tumors, and decreased expression of AR was seen in 63% (22 of 35) of tumors. Cell type and stage did not influence differential expression, indicating that this is a frequent event in primary NSCLC. Simultaneous overexpression of EGFR and TGF-alpha was seen in only 38% of tumors. Simultaneous overexpression of EGFR and decreased expression of AR were seen in only 21% of tumors. Thus far, the differential expression of EGFR, TGF-alpha, and AR does not correlate with either disease-free or overall survival. These findings indicate that histologically dissimilar tumors can express similar components of autocrine or paracrine growth factor loops. Differential expression of EGFR and its ligands in tumor specimens compared to uninvolved lung is a common event in NSCLC and may participate in tumor growth without necessarily influencing tumor progression or histology.

Wavelength Dependence of Skin Cancer Induction by Ultraviolet Irradiation of Albino Hairless Mice

Abstract: Information on the variation in carcinogenicity with wavelength is crucial in risk assessments for skin cancers induced by UV radiation. Until recently the wavelength (lambda) dependencies of other detrimental UV effects, such as sunburn, have been used as substitutes. Direct information on the lambda dependency can only be obtained from animal experiments. To this end we accumulated a large data set on skin tumors induced by chronic UV exposure of albino SKH:HR1 mice (14 different broadband UV sources and about 1100 mice); the data come from the Photobiology Unit of the former Skin and Cancer Hospital in Philadelphia and from the Department of Dermatology of the University of Utrecht. The lambda dependency was extracted from this data set (a statistically satisfactory description with chi 2 = 13.4, df = 7) and represented by the Skin Cancer Utrecht-Philadelphia action spectrum, i.e., a set of factors to weight the exposures at different wavelengths according to their respective effectiveness (inversely proportional to the daily exposure required for a median tumor induction time of 300 days). The fits obtained with other already available action spectra proved to be poor (chi 2 > 60, df = 11). The maximum effectiveness was found at 293 nm, and above 340 nm the effectiveness showed a shoulder at about 10(-4) of the maximum. A sensitivity analysis of the final solution for the lambda dependency showed a large margin of uncertainty above 340 nm and an information gap below 280 nm. The large variation in tumor responses in the present data set can be transformed to a coherent, common dose-response relationship by proper spectral weighting with this single action spectrum.

Genetic instability in pancreatic cancer and poorly differentiated type of gastric cancer.

Abstract: To examine genetic instability during carcinogenesis, we screened 171 carcinomas of the breast, liver, proximal colon, stomach, pancreas, uterine cervix, and ovary for replication error at four microsatellite marker loci on chromosome 2, 3, and 17. A significantly high incidence of genetic instability was observed in pancreatic (6 of 9 tumors) and gastric cancers (22 of 57 cases). In other types of carcinoma, the incidence of replication error-positive cases was relatively low (3-16%). Among gastric carcinomas, significantly more replication error-positive cases were observed in the poorly differentiated types (16 of 25 cases) than in well differentiated types (3 of 18) (P = 0.0023 by Fisher's exact test). Our results suggested that genetic instability is likely to play an important role in development of pancreatic and gastric cancers, particularly poorly differentiated adenocarcinomas.

Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease.

Abstract: The HER-2/neu proto-oncogene (also known as c-erb B-2) is homologous with, but distinct from, the epidermal growth factor receptor. Amplification of this gene in node-positive breast cancers has been shown to correlate with both earlier relapse and shorter overall survival. In node-negative breast cancer patients, the subgroup for which accurate prognostic data could make a significant contribution to treatment decisions, the prognostic utility of HER-2/neu amplification and/or overexpression has been controversial. The purpose of this report is to address the issues surrounding this controversy and to evaluate the prognostic utility of overexpression in a carefully followed group of patients using appropriately characterized reagents and methods. In this report we present data from a study of HER-2/neu expression designed specifically to test whether or not overexpression is associated with an increased risk of recurrence in node-negative breast cancers. From a cohort of 704 women with node-negative breast cancer who experienced recurrent disease (relapsed cases) 105 were matched with 105 women with no recurrence (disease-free controls) after the equivalent follow-up period. Immunohistochemistry was used to assess HER-2/neu expression in archival tissue blocks from both relapsed cases and their matched disease-free controls. Importantly, a series of molecularly characterized breast cancer specimens were used to confirm that the antibody used was of sufficient sensitivity and specificity to identify those cancers overexpressing the HER-2/neu protein in this formalin-fixed, paraffin-embedded tissue cohort. In addition, a quantitative approach was developed to more accurately assess the amount of HER-2/neu protein identified by immunostaining tumor tissue. This was done using a purified HER-2/neu protein synthesized in a bacterial expression vector and protein lysates derived from a series of cell lines, engineered to express a defined range of HER-2/neu oncoprotein levels. By using cells with defined expression levels as calibration material, computerized image analysis of immunohistochemical staining could be used to determine the amount of oncoprotein product in these cell lines as well as in human breast cancer specimens. Quantitation of the amount of HER-2/neu protein product determined by computerized image analysis of immunohistochemical assays correlated very closely with quantitative analysis of a series of molecularly characterized breast cancer cell lines and breast cancer tissue specimens.(ABSTRACT TRUNCATED AT 400 WORDS)

Antitumor Effect of Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies plus cis-Diamminedichloroplatinum on Well Established A431 Cell Xenografts

Abstract: We have explored the therapeutic effects of anti-epidermal growth factor receptor monoclonal antibodies (MAbs) 225 and 528 on well established A431 epidermoid carcinoma xenografts, approximately 400 mm3 (1 cm in diameter) at the start of treatment. In previous reports we demonstrated that MAbs 225 and 528 prevented the growth of A431 cell xenografts in nude mice when treatment was begun on the day of tumor cell inoculation. Since anti-epidermal growth factor receptor MAb therapy of well established tumors was unable to retard growth, we explored combination therapy with MAb plus the chemotherapeutic agent cis-diamminedichloroplatinum (cis-DDP). Additive and concentration-dependent growth-inhibitory effects of MAb with cis-DDP were observed in cultures of A431 cells. Neither intensive treatment with 225 MAb (1 mg/mouse, i.p. on day 8 after tumor inoculation, and twice weekly for 4 weeks) nor a maximally tolerated single dose of cis-DDP [150 micrograms/25 g (6 mg/kg) mouse weight, i.p. on day 8] had significant effects on tumor growth. However, the two treatments in combination resulted in substantial xenograft growth inhibition, compared with both an untreated control group and animals treated with a single modality. When a second dose of cis-DDP (150 micrograms/25 g) was added after 10 days, combination therapy with 225 MAb produced striking antitumor effects. At the end of 1 month tumor xenografts had disappeared in all but one mouse, and no tumor relapses occurred during 6 months of observation. Identical results were obtained with anti-epidermal growth factor receptor MAb 528 in combination with cis-DDP. The results of these studies provide a novel approach to the treatment of well established tumor xenografts, which may have application in the therapy of human malignancies.

Levels of Matrix Metalloproteases in Bladder Cancer Correlate with Tumor Grade and Invasion

Abstract: We have used quantitative zymography to measure levels of the type IV collagenases matrix metalloproteinase (MMP)-9 and MMP-2 in 42 biopsies of transitional cell carcinoma and in 7 biopsies of normal bladder. Mean levels of MMP-9 were significantly higher in tumor compared with normal samples ( P = 0.08). Levels of MMP-9 and active MMP-2 increased with tumor grade (test for trend, P = 0.002 and P = 0.05, respectively). Levels of MMP-9 and activated MMP-2 were also higher in invasive tumors than in superficial ones ( P = 0.001 and P = 0.008, respectively). In situ hybridization studies showed that the mRNAs for both MMP-2 and MMP-9 were located chiefly in the stroma rather than epithelial tumor cells and were concentrated at the interface between the two tissues.