Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells

Epithelial (E)-cadherin and its associated cytoplasmic proteins (α-, β-, and γ-catenins) are important mediators of epithelial cell–cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin–catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.

Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

The functional role of the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in epidermal growth factor (EGF)-induced expression of 12(S)-lipoxygenase gene in human epidermoid carcinoma A431 cells was studied. Coimmunoprecipitation experiments indicated that EGF stimulated interaction between c-Jun and Sp1 in a time-dependent manner. Overexpression of Ha-ras and c-Jun also enhanced the amount of c-Jun binding to Sp1. In addition, the c-Jun dominant negative mutant TAM-67 not only inhibited the coimmunoprecipitated c-Jun binding to Sp1 in a dose-dependent manner in cells overexpressing c-Jun but also reduced promoter activity of the 12(S)-lipoxygenase gene induced by c-Jun overexpression. Treatment of cells with EGF increased the interaction between the Sp1 oligonucleotide and nuclear c-Jun/Sp1 in a time-dependent manner. Furthermore, EGF activated the chimeric promoter consisting of 10 tandem GAL4-binding sites, which replaced the three Sp1-binding sites in the 12(S)lipoxygenase promoter only when coexpressed with GAL4-c-Jun () fusion proteins. These results indicate that the direct interaction between c-Jun and Sp1 induced by EGF cooperatively activated expression of the 12(S)-lipoxygenase gene, and that Sp1 may serve at least in part as a carrier bringing c-Jun to the promoter, thus transactivating the transcriptional activity of 12(S)-lipoxygenase gene.

Formation of stable adducts and absence of depurinating DNA adducts in cells and DNA treated with the potent carcinogen dibenzo[a,l]pyrene or its diol epoxides

Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants, and some are potent carcinogens in rodents. Carcinogenic PAH are activated in cells to metabolites that react with DNA to form stable covalent DNA adducts. It has been proposed [Cavalieri, E. L. & Roger, E. G. (1995) Xenobiotica 25, 677–688] that unstable DNA adducts are also formed and that apurinic sites in the DNA resulting from unstable PAH adducts play a key role in the initiation of cancer. The potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) is activated in cells to (+)-syn- and (−)-anti-DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE), which have been shown to form stable adducts with DNA. To evaluate the importance of unstable PAH adducts, we compared stable adduct formation to apurinic site formation. Stable DB[a,l]PDE adducts were determined by 33P-postlabeling and HPLC. To measure apurinic sites they were converted to strand breaks, and these were monitored by examining the integrity of a particular restriction fragment of the dihydrofolate reductase gene. The method easily detected apurinic sites resulting from methylation by treatment of cells or DNA with dimethyl sulfate or from reaction of DNA with DB[a,l]P in the presence of horseradish peroxidase. We estimate the method could detect 0.1 apurinic site in the 14-kb fragment examined. However, apurinic sites were below our limit of detection in DNA treated directly with (+)-syn- or (−)-anti-DB[a,l]PDE or in DNA from Chinese hamster ovary B11 cells so treated, although in these samples the frequency of stable adducts ranged from 3 to 10 per 14 kb. We also treated the human mammary carcinoma cell line MCF-7 with DB[a,l]P and again could not detect significant amounts of unstable adducts. These results indicate that the proportion of stable adducts formed by DB[a,l]P activated in cells and its diol epoxides is greater than 99% and suggest a predominant role for stable DNA adducts in the carcinogenic activity of DB[a,l]P.

Distinct roles for the N- and C-terminal regions in the cytotoxicity of pierisin-1, a putative ADP-ribosylating toxin from cabbage butterfly, against mammalian cells

Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage butterfly, Pieris rapae, and has been suggested to consist of an N-terminal region with ADP-ribosyltransferase domain and of a C-terminal region that might have a receptor-binding domain. To elucidate the role of each region, we investigated the functions of various fragments of pierisin-1. In vitro expressed polypeptide consisting of amino acid residues 1–233 or 234–850 of pierisin-1 alone did not show cytotoxicity against human cervical carcinoma HeLa cells. However, the presence of both polypeptides in the culture medium showed some of the original cytotoxic activity. Introduction of the N-terminal polypeptide alone by electroporation also induced cell death in HeLa cells, and even in the mouse melanoma MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a principal role in the cytotoxicity of pierisin-1 inside mammalian cells. Analyses of incorporated pierisin-1 indicated that the entire protein, regardless of whether it consisted of a single polypeptide or two separate N- and C-terminal polypeptides, was incorporated into HeLa cells. However, neither of the terminal polypeptides was incorporated when each polypeptide was present separately. These findings indicate that the C-terminal region is important for the incorporation of pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid fraction of cell membrane was suggested. The cytotoxic effects of pierisin-1 were enhanced by previous treatment with trypsin, producing “nicked” pierisin-1. Generation of the N-terminal fragment in HeLa cells was detected after application of intact entire molecule of pierisin-1. From the above observations, it is suggested that after incorporation of pierisin-1 into the cell by interaction of its C-terminal region with the receptor in the cell membrane, the entire protein is cleaved into the N- and C-terminal fragments with intracellular protease, and the N-terminal fragment then exhibits cytotoxicity.

The synthesis, discovery, and development of a highly promising class of microtubule stabilization agents: Curative effects of desoxyepothilones B and F against human tumor xenografts in nude mice

We have evaluated two synthetic epothilone analogues lacking the 12,13-epoxide functionality, 12,13-desoxyepothilone B (dEpoB), and 12,13-desoxyepothilone F (dEpoF). The concentrations required for 50% growth inhibition (IC50) for a variety of anticancer agents were measured in CCRF-CEM/VBL1000 cells (2,048-fold resistance to vinblastine). By using dEpoB, dEpoF, aza-EpoB, and paclitaxel, the IC50 values were 0.029, 0.092, 2.99, and 5.17 μM, respectively. These values represent 4-, 33.5-, 1,423- and 3,133-fold resistance, respectively, when compared with the corresponding IC50 in the parent [nonmultiple drug-resistant (MDR)] CCRF-CEM cells. We then produced MDR human lung carcinoma A549 cells by continuous exposure of the tumor cells to sublethal concentrations of dEpoB (1.8 yr), vinblastine (1.2 yr), and paclitaxel (1.8 yr). This continued exposure led to the development of 2.1-, 4,848-, and 2,553-fold resistance to each drug, respectively. The therapeutic effect of dEpoB and paclitaxel was also compared in vivo in a mouse model by using various tumor xenografts. dEpoB is much more effective in reducing tumor sizes in all MDR tumors tested. Analysis of dEpoF, an analog possessing greater aqueous solubility than dEpoB, showed curative effects similar to dEpoB against K562, CCRF-CEM, and MX-1 xenografts. These results indicate that dEpoB and dEpoF are efficacious antitumor agents with both a broad chemotherapeutic spectrum and wide safety margins.

Human prostate tumor growth in athymic mice: inhibition by androgens and stimulation by finasteride.

When the human prostate cancer cell line, LNCaP 104-S, the growth of which is stimulated by physiological levels of androgen, is cultured in androgen-depleted medium for > 100 passages, the cells, now called LNCaP 104-R2, are proliferatively repressed by low concentrations of androgens. LNCaP 104-R2 cells formed tumors in castrated male athymic nude mice. Testosterone propionate (TP) treatment prevented LNCaP 104-R2 tumor growth and caused regression of established tumors in these mice. Such a tumor-suppressive effect was not observed with tumors derived from LNCaP 104-S cells or androgen receptor-negative human prostate cancer PC-3 cells. 5 alpha-Dihydrotestosterone, but not 5 beta-dihydrotestosterone, 17 beta-estradiol, or medroxyprogesterone acetate, also inhibited LNCaP 104-R2 tumor growth. Removal of TP or implantation of finasteride, a 5 alpha-reductase inhibitor, in nude mice bearing TP implants resulted in the regrowth of LNCaP 104-R2 tumors. Within 1 week after TP implantation, LNCaP 104-R2 tumors exhibited massive necrosis with severe hemorrhage. Three weeks later, these tumors showed fibrosis with infiltration of chronic inflammatory cells and scattered carcinoma cells exhibiting degeneration. TP treatment of mice with LNCaP 104-R2 tumors reduced tumor androgen receptor and c-myc mRNA levels but increased prostate-specific antigen in serum- and prostate-specific antigen mRNA in tumors. Although androgen ablation has been the standard treatment for metastatic prostate cancer for > 50 years, our study shows that androgen supplementation therapy may be beneficial for treatment of certain types of human prostate cancer and that the use of 5 alpha-reductase inhibitors, such as finasteride or anti-androgens, in the general treatment of metastatic prostate cancer may require careful assessment.

Interleukin 7 independent development of human B cells.

Mammalian hematopoietic stem cell (HSC) commitment and differentiation into lymphoid lineage cells proceed through a series of developmentally restricted progenitor compartments. A complete understanding of this process, and how it differs from HSC commitment and differentiation into cells of the myeloid/erythroid lineages, requires the development of model systems that support HSC commitment to the lymphoid lineages. We now describe a human bone marrow stromal cell culture that preferentially supports commitment and differentiation of human HSC to CD19+ B-lineage cells. Fluorescence activated cell sorterpurified CD34++/lineage-cells were isolated from fetal bone marrow and cultured on human fetal bone marrow stromal cells in serum-free conditions containing no exogenous cytokines. Over a period of 3 weeks, CD34++/lineage- cells underwent commitment, differentiation, and expansion into the B lineage. Progressive changes included: loss of CD34, acquisition of and graded increases in the level of cell surface CD19, and appearance of immature B cells expressing mu/kappa or mu/lambda cell surface Ig receptors. The tempo and phenotype of B-cell development was not influenced by the addition of IL-7 (10 ng/ml), or by the addition of goat anti-IL-7 neutralizing antibody. These results indicate a profound difference between mouse and human in the requirement for IL-7 in normal B-cell development, and provide an experimental system to identify and characterize human bone marrow stromal cell-derived molecules crucial for human B lymphopoiesis.

Specific accumulation of tumor-derived adhesion factor in tumor blood vessels and in capillary tube-like structures of cultured vascular endothelial cells.

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Antibody-directed enzyme prodrug therapy, ADEPT, is a recent approach to targeted cancer chemotherapy intended to diminish the nonspecific toxicity associated with many commonly used chemotherapeutic agents. Most ADEPT systems incorporate a bacterial enzyme, and thus their potential is reduced because of the immunogenicity of that component of the conjugate. This limitation can be circumvented by the use of a catalytic antibody, which can be "humanized," in place of the bacterial enzyme catalyst. We have explored the scope of such antibody-directed "abzyme" prodrug therapy, ADAPT, to evaluate the potential for a repeatable targeted cancer chemotherapy. We report the production of a catalytic antibody that can hydrolyze the carbamate prodrug 4-[N,N-bis(2-chloroethyl)]aminophenyl-N-[(1S)-(1,3- dicarboxy)propyl]carbamate (1) to generate the corresponding cytotoxic nitrogen mustard (Km = 201 microM, kcat = 1.88 min-1). In vitro studies with this abzyme, EA11-D7, and prodrug 1 lead to a marked reduction in viability of cultured human colonic carcinoma (LoVo) cells relative to appropriate controls. In addition, we have found a good correlation between antibody catalysis as determined by this cytotoxicity assay in vitro and competitive binding studies of candidate abzymes to the truncated transition-state analogue ethyl 4-nitrophenylmethylphosphonate. This cell-kill assay heralds a general approach to direct and rapid screening of antibody libraries for catalysts.

CD4+ blood lymphocytes are rapidly killed in vitro by contact with autologous human immunodeficiency virus-infected cells.

We have investigated the ability of human immunodeficiency virus (HIV)-infected cells to kill uninfected CD4+ lymphocytes. Infected peripheral blood mononuclear cells were cocultured with autologous 51Cr-labeled uninfected cells. Rapid death of the normal CD4-expressing target population was observed following a brief incubation. Death of blood CD4+ lymphocytes occurred before syncytium formation could be detected or productive viral infection established in the normal target cells. Cytolysis could not be induced by free virus, was dependent on gp120-CD4 binding, and occurred in resting, as well as activated, lymphocytes. CD8+ cells were not involved in this phenomenon, since HIV-infected CEMT4 cells (CD4+, CD8- cells) mediated the cytolysis of uninfected targets. Reciprocal isotope-labeling experiments demonstrated that infected CEMT4 cells did not die in parallel with their targets. The uninfected target cells manifested DNA fragmentation, followed by the release of the 51Cr label. Thus, in HIV patients, infected lymphocytes may cause the depletion of the much larger population of uninfected CD4+ cells without actually infecting them, by triggering an apoptotic death.

Interferons alpha and beta down-regulate the expression of basic fibroblast growth factor in human carcinomas.

We investigated the influence of interferons alpha, beta, and gamma (IFN-alpha, -beta, and -gamma) on the production of basic fibroblast growth factor (bFGF) by human renal carcinoma cells. The human renal carcinoma cell metastatic line SN12PM6 was established in culture from a lung metastasis and SN12PM6-resistant cells were selected in vitro for resistance to the antiproliferative effects of IFN-alpha or IFN-beta. IFN-alpha and IFN-beta, but not IFN-gamma, down-regulated the expression of bFGF at the mRNA and protein levels by a mechanism independent of their antiproliferative effects. Down-regulation of bFGF required a long exposure (> 4 days) of cells to low concentrations (> 10 units/ml) of IFN-alpha or IFN-beta. The withdrawal of IFN-alpha or IFN-beta from the medium permitted SN12PM6-resistant cells to resume production of bFGF. The incubation of human bladder, prostate, colon, and breast carcinoma cells with noncytostatic concentrations of IFN-alpha or IFN-beta also produced down-regulation of bFGF production.

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 → S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: A possible mechanism of breast carcinogenesis

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G2 cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.

Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity

Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1S12OG and nm23-H1P96S. Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1P96S or nm23-H1S120G, and another was phosphorylated much more slowly by nm23-H1P96S and by nm23-H1S120G than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATP-citrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1S120G and nm23-H1P96S were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1S120 was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1P96S was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.

Pig but not human interferon-γ initiates human cell-mediated rejection of pig tissue in vivo

Split-thickness pig skin was transplanted on severe combined immunodeficient mice so that pig dermal microvessels spontaneously inosculated with mouse microvessels and functioned to perfuse the grafts. Pig endothelial cells in the healed grafts constitutively expressed class I and class II major histocompatibility complex molecules. Major histocompatibility complex molecule expression could be further increased by intradermal injection of pig interferon-γ (IFN-γ) but not human IFN-γ or tumor necrosis factor. Grafts injected with pig IFN-γ also developed a sparse infiltrate of mouse neutrophils and eosinophils without evidence of injury. Introduction of human peripheral blood mononuclear cells into the animals by intraperitoneal inoculation resulted in sparse perivascular mononuclear cell infiltrates in the grafts confined to the pig dermis. Injection of pig skin grafts on mice that received human peripheral blood mononuclear cells with pig IFN-γ (but not human IFN-γ or heat-inactivated pig IFN-γ) induced human CD4+ and CD8+ T cells and macrophages to more extensivley infiltrate the pig skin grafts and injure pig dermal microvessels. These findings suggest that human T cell-mediated rejection of xenotransplanted pig organs may be prevented if cellular sources of pig interferon (e.g., passenger lymphocytes) are eliminated from the graft.