Showing papers on "Cancer cell published in 1997"

CD44: Structure, Function and Association with the Malignant Process

Abstract: CD44 is a ubiquitous multistructural and multifunctional cells surface adhesion molecule involved in cell-cell and cell-matrix interactions. Twenty exons are involved in the genomic organization of this molecule. The first five and the last 5 exons are constant, whereas the 10 exons located between these regions are subjected to alternative splicing, resulting in the generation of a variable region. Differential utilization of the 10 variable region exons, as well as variations in N-glycosylation, O-glycosylation, and glycosaminoglycanation (by heparan sulfate or chondroitin sulfate), generate multiple isoforms (at least 20 are known) of different molecular sizes (85-230 kDa). The smallest CD44 molecule (85-95 kDa), which lacks the entire variable region, is standard CD44 (CD44s). As it is expressed mainly on cells of lymphohematopoietic origin, CD44s is also known as hematopoietic CD44 (CD44H). CD44s is a single-chain molecule composed of a distal extracellular domain (containing, the ligand-binding sites), a membrane-proximal region, a transmembrane-spanning domain, and a cytoplasmic tail. The molecular sequence (with the exception of the membrane-proximal region) displays high interspecies homology. After immunological activation, T lymphocytes and other leukocytes transiently upregulate CD44 isoforms expressing variant exons (designated CD44v). A CD44 isform containing the last 3 exon products of the variable region (CD44V8-10, also known as epithelial CD44 or CD44E), is preferentially expressed on epithelial cells. The longest CD44 isoform expressing in tandem eight exons of the variable region (CD44V3-10) was detected in keratinocytes. Hyaluronic acid (HA), an important component of the extracellular matrix (ECM), is the principal, but by no means the only, ligand of CD44. Other CD44 ligands include the ECM components collagen, fibronectin, laminin, and chondroitin sulfate. Mucosal addressin, serglycin, osteopontin, and the class II invariant chain (Ii) are additional, ECM-unrelated, ligands of the molecule. In many, but not in all cases, CD44 does not bind HA unless it is stimulated by phorbol esters, activated by agonistic anti-CD44 antibody, or deglycosylated (e.g., by tunicamycin). CD44 is a multifunctional receptor involved in cell-cell and cell-ECM interactions, cell traffic, lymph node homing, presentation of chemokines and growth factors to traveling cells, and transmission of growth signals. CD44 also participates in the uptake and intracellular degradation of HA, as well as in transmission of signals mediating hematopoiesis and apoptosis. Many cancer cell types as well as their metastases express high levels of CD44. Whereas some tumors, such as gliomas, exclusively express standard CD44, other neoplasms, including gastrointestinal cancer, bladder cancer, uterine cervical cancer, breast cancer and non-Hodgkin's lymphomas, also express CD44 variants. Hence CD44, particularly its variants, may be used as diagnostic or prognostic markers of at least some human malignant diseases. Furthermore, it has been shown in animal models that injection of reagents interfering with CD44-ligand interaction (e.g., CD44s- or CD44v-specific antibodies) inhibit local tumor growth and metastatic spread. These findings suggest that CD44 may confer a growth advantage on some neoplastic cells and, therefore, could be used as a target for cancer therapy. It is hoped that identification of CD44 variants expressed on cancer but not on normal cells will lead to the development of anti-CD44 reagents restricted to the neoplastic growth.

Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins

Abstract: The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [mdr1b (−/−) mice] and in both the mdr1a and mdr1b genes [mdr1a/1b (−/−) mice]. In spite of the host of functions speculatively attributed to the mdr1-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum–chemical, and immunological parameters were not abnormal in mdr1a/1b (−/−) mice. The high level of mdr1b P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically, mdr1a/1b (−/−) mice behaved similarly to the previously analyzed mdr1a (−/−) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability of mdr1a/1b (−/−) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy. mdr1a/1b (−/−) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.

c-Myc transactivation of LDH-A: Implications for tumor metabolism and growth

Abstract: Cancer cells are able to overproduce lactic acid aerobically, whereas normal cells undergo anaerobic glycolysis only when deprived of oxygen. Tumor aerobic glycolysis was recognized about seven decades ago; however, its molecular basis has remained elusive. The lactate dehydrogenase-A gene (LDH-A), whose product participates in normal anaerobic glycolysis and is frequently increased in human cancers, was identified as a c-Myc-responsive gene. Stably transfected Rat1a fibroblasts that overexpress LDH-A alone or those transformed by c-Myc overproduce lactic acid. LDH-A overexpression is required for c-Myc-mediated transformation because lowering its level through antisense LDH-A expression reduces soft agar clonogenicity of c-Myc-transformed Rat1a fibroblasts, c-Myc-transformed human lymphoblastoid cells, and Burkitt lymphoma cells. Although antisense expression of LDH-A did not affect the growth of c-Myc-transformed fibroblasts adherent to culture dishes under normoxic conditions, the growth of these adherent cells in hypoxia was reduced. These observations suggest that an increased LDH-A level is required for the growth of a transformed spheroid cell mass, which has a hypoxic internal microenvironment. Our studies have linked c-Myc to the induction of LDH-A, whose expression increases lactate production and is necessary for c-Myc-mediated transformation.

c-Myc transactivation of LDH-A: Implications for tumor metabolism and growth (oncogeneylactate dehydrogenaseyhypoxiaytumorigenicity)

Abstract: Cancer cells are able to overproduce lactic acid aerobically, whereas normal cells undergo anaerobic glycolysis only when deprived of oxygen. Tumor aerobic glycolysis was recognized about seven decades ago; however, its molecular basis has remained elusive. The lactate dehy- drogenase-A gene (LDH-A), whose product participates in normal anaerobic glycolysis and is frequently increased in human cancers, was identified as a c-Myc-responsive gene. Stably transfected Rat1a fibroblasts that overexpress LDH-A alone or those transformed by c-Myc overproduce lactic acid. LDH-A overexpression is required for c-Myc-mediated trans- formation because lowering its level through antisense LDH-A expression reduces soft agar clonogenicity of c-Myc- transformed Rat1a fibroblasts, c-Myc-transformed human lymphoblastoid cells, and Burkitt lymphoma cells. Although antisense expression of LDH-A did not affect the growth of c-Myc-transformed fibroblasts adherent to culture dishes under normoxic conditions, the growth of these adherent cells in hypoxia was reduced. These observations suggest that an increased LDH-A level is required for the growth of a trans- formed spheroid cell mass, which has a hypoxic internal microenvironment. Our studies have linked c-Myc to the induction of LDH-A, whose expression increases lactate pro- duction and is necessary for c-Myc-mediated transformation.

Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth.

Abstract: Breast cancers often progress from a hormone-dependent, nonmetastatic, antiestrogen-sensitive phenotype to a hormone-independent, antiestrogen- and chemotherapy-resistant phenotype with highly invasive and metastatic growth properties. This progression is usually accompanied by altered function of the estrogen receptor (ER) or outgrowth of ER-negative cancer cells. To understand the molecular mechanisms responsible for metastatic growth of ER-negative breast cancers, the activities of the transcription factor NF-kappaB (which modulates the expression of genes involved in cell proliferation, differentiation, apoptosis, and metastasis) were compared in ER-positive (MCF-7 and T47-D) and ER-negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines. NF-kappaB, which is usually maintained in an inactive state by protein-protein interaction with inhibitor IkappaBs, was found to be constitutively active in ER-negative breast cancer cell lines. Constitutive DNA binding of NF-kappaB was also observed with extracts from ER-negative, poorly differentiated primary breast tumors. Progression of the rat mammary carcinoma cell line RM22-F5 from an ER-positive, nonmalignant phenotype (E phenotype) to an ER-negative, malignant phenotype (F phenotype) was also accompanied by constitutive activation of NF-kappaB. Analysis of individual subunits of NF-kappaB revealed that all ER-negative cell lines, including RM22-F5 cells of F phenotype, contain a unique 37-kDa protein which is antigenically related to the RelA subunit. Cell-type-specific differences in IkappaB alpha, -beta, and -gamma were also observed. In transient-transfection experiments, constitutive activity of an NF-kappaB-dependent promoter was observed in MDA-MB-231 and RM22-F5 cells of F phenotype, and this activity was efficiently repressed by cotransfected ER. Since ER inhibits the constitutive as well as inducible activation function of NF-kappaB in a dose-dependent manner, we propose that breast cancers that lack functional ER overexpress NF-kappaB-regulated genes. Furthermore, since recent data indicate that NF-kappaB protects cells from tumor necrosis factor alpha-, ionizing radiation-, and chemotherapeutic agent daunorubicin-mediated apoptosis, our results provide an explanation for chemotherapeutic resistance in ER-negative breast cancers.

Bcl2 Is the Guardian of Microtubule Integrity

Abstract: We have investigated the ability of several drugs commonly used in the treatment of human cancer to induce bcl2 phosphorylation and cell death in human cell lines derived from acute leukemia, lymphoma, breast cancer, and prostate cancer. The results of this analysis indicate that drugs affecting the integrity of microtubules induce bc12 phosphorylation, whereas anticancer drugs damaging DNA do not. Comparison of the effects of taxol and its analogue, taxotere, indicates that taxotere is capable of inducing bcl2 phosphorylation and apoptotic cell death at 100-fold lower concentrations than taxol. Induction of cancer cell death through phosphorylation of bcl2 thus provides an opportunity not only for more refined targeting of therapeutic drugs but for understanding of an important pathway leading to apoptosis. Phosphorylation of bcl2 in drug-treated cancer cells occurs in G2-M, the phase of the cell cycle in which this class of drugs is active. No induction of bcl2 phosphorylation occurs in chronic lymphocytic leukemia cells that overexpress bcl2 but are blocked at G0-G1. Thus, prevention of polymerization or depolymerization of cellular microtubules by this class of cancer therapeutic drugs causes phosphorylation of bcl2, abrogating the normal antiapoptotic function of bcl2 and initiating the apoptotic program in the cycling cancer cells; these results are consistent with a normal physiological role of bcl2 as "guardian of microtubule integrity."

Clinical impact of the plasminogen activation system in tumor invasion and metastasis : Prognostic relevance and target for therapy

Abstract: Extravasation and intravasation of solid malignant tumors is controlled by attachment of tumor cells to components of the basement membrane and the extracellular matrix, by local proteolysis and tumor cell migration. Strong clinical and experimental evidence has accumulated that the tumor-associated serine protease plasmin, its activator uPA (urokinase-type plasminogen activator), the receptor uPA-R (CD87), and the inhibitors PAI-1 and PAI-2 are linked to cancer invasion and metastasis. In cancer, increase of uPA, uPA-R, and/or PAI-1 is associated with tumor progression and with shortened disease-free and/or overall survival in patients afflicted with malignant solid tumors. uPA and/or its inhibitor PAI-1 appear to be one of the strongest prognostic markers so far described. Strong prognostic value to predict disease recurrence and overall survival has been documented for patients with cancer of the breast, ovary, cervix, endometrium, stomach, colon, lung, bladder, kidney, brain, and soft-tissue. Due to the strong correlation between elevated uPA and/or PAI-1 values in primary cancer tissues and the tumor invasion/ metastasis capacity of cancer cells, proteolytic factors have been selected as targets for therapy. Various very different approaches to interfere with the expression or reactivity of uPA or CD87 at the gene or protein level were successfully tested including antisense oligonucleotides, antibodies, enzyme inhibitors, and recombinant or synthetic uPA and uPA-R analogues.

DNA methylation and genetic instability in colorectal cancer cells

Abstract: Apparent alterations in DNA methylation have been observed in many cancers, but whether such alterations represent a persistent alteration in the normal methylation process is not known. In this study, we report a striking difference in the expression of exogenously introduced retroviral genes in various colorectal cancer cell lines. Extinguished expression was associated with DNA methylation and could be reversed by treatment with the demethylating agent 5-azacytidine. A striking correlation between genetic instability and methylation capacity suggested that methylation abnormalities may play a role in chromosome segregation processes in cancer cells.

High level expression of p27(kip1) and cyclin D1 in some human breast cancer cells: inverse correlation between the expression of p27(kip1) and degree of malignancy in human breast and colorectal cancers.

Abstract: The expression of cyclin-dependent kinase inhibitor p27kip1 in human tumors and normal tissues was investigated using a panel of novel anti-p27kip1 mAbs An inverse correlation between expression of p27kip1 and cell proliferation was generally observed after analyzing its expression in 25 different normal human tissues In some highly proliferative human breast cancer cells, however, high level p27kip1 expression was seen, indicating the existence of a mechanism by which some growing tumor cells may tolerate this inhibitor of cell cycle progression Detailed studies demonstrated a correlation between the high level expression of p27kip1 and cyclin D1 in human breast cancer cells There was also an inverse correlation between the expression of p27kip1 and the degree of tumor malignancy in human breast and colorectal cancers, indicating that p27kip1 may be a useful prognostic marker in these cancers

Antioxidant enzyme levels in cancer.

Abstract: Normal cells are protected by antioxidant enzymes from the toxic effects of high concentrations of reactive oxygen species generated during cellular metabolism. Even though cancer cells generate reactive oxygen species, it has been demonstrated biochemically that antioxidant enzyme levels are low in most animal and human cancers. However, a few cancer types have been found to have elevated levels of antioxidant enzymes, particularly manganese superoxide dismutase. Morphologic studies of animal and human cancer have confirmed that although the majority of tumor cell types from several organ systems have low antioxidant enzymes, adenocarcinomas may have elevated manganese superoxide dismutase and catalase levels. However, all cancers examined to date have some imbalance in antioxidant enzyme levels compared with the cell of origin. Antioxidant enzyme importance in cancer genesis has been difficult to evaluate in early cancerous lesions using biochemical techniques because such lesions are small and therefore below the level of detection. Using immunohistochemical techniques, early lesions of human and animal cancers were demonstrated to have low antioxidant enzymes, thus suggesting a role for these enzymes both in the genesis of cancer and the malignant phenotype. All but one human cancer cell type (the granular cell variant of human renal adenocarcinoma) examined showed both low catalase and glutathione peroxidase levels, suggesting that most cancer cell types cannot detoxify hydrogen peroxide. Our results to date are used to propose new cancer therapies based on modulation of cellular redox state.

Integrins, oncogenes, and anchorage independence

Abstract: The ability of cancer cells to proliferate in the absence of adhesion to extracellular matrix (ECM)1 proteins, termed anchorage independence of growth, correlates closely with tumorigenicity in animal models (14). This property of cancer cells presumably reflects the tendency of tumor cells to survive and grow in inappropriate locations in vivo. Such incorrect localization, as occurs in invasion and metastasis, is the characteristic that distinguishes malignant from benign tumors (31). Great progress has been made in the last 20 years toward understanding how growth is controlled in normal cells and how oncogenes usurp these controls. Yet studies on how oncogenes (or loss of tumor suppressors) overcome the mechanisms that govern cellular location have lagged considerably. The finding that integrins transduce signals that influence intracellular growth regulatory pathways provided some insight into anchorage dependence. Available evidence indicates that integrin-dependent signals mediate the growth requirement for cell adhesion to ECM proteins. Our understanding of integrin signaling has now reached a stage that connections to oncogenesis are becoming clear, enabling us to place a number of proto-oncogenes and oncogenes with respect to their adhesion dependence or independence. While many details of molecular mechanisms remain to be elucidated, sufficient information is now available to propose a general framework for how oncogenes lead to anchorage-independent growth.

Carbohydrate-mediated cell adhesion involved in hematogenous metastasis of cancer

Abstract: The carbohydrate determinants, sialyl Lewis A and sialyl Lewis X, which are frequently expressed on human cancer cells, serve as ligands for a cell adhesion molecule of the selectin family, E-selectin, which is expressed on vascular endothelial cells. These carbohydrate determinants are involved in the adhesion of cancer cells to vascular endothelium and thus contribute to hematogenous metastasis of cancer. The initial adhesion mediated by these molecules triggers activation of integrin molecules through the action of several cytokines and leads to the extravasation of cancer cells. Cancer cells also produce humoral factors that facilitate E-selectin expression on endothelial cells. The degree of expression of the carbohydrate ligands at the surface of cancer cells is well correlated with the frequency of hematogenous metastasis and prognostic outcome of patients with cancers. The alteration of glycosyltransferase activities that leads to the enhanced expression of these carbohydrate ligands on cancer cell surface are currently being investigated.

Apoptosis, cancer and cancer therapy

Abstract: Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis. Apoptosis is essential in the homeostasis of normal tissues of the body, especially those of the gastrointestinal tract, immune system and skin. There is increasing evidence that the processes of neoplastic transformation, progression and metastasis involve alterations in the normal apoptotic pathways. Furthermore, the majority of chemotherapeutic agents as well as radiation utilize the apoptotic pathway to induce cancer cell death. Resistance to standard chemotherapies also seems to be determined by alterations in the apoptotic pathways of cancer cells. Therefore, understanding the signals of apoptosis and the mechanism of apoptosis may allow the development of better chemo- or radiotherapeutic regimens for the treatment of cancer. Finally, components of the apoptotic pathway may represent potential therapeutic targets using gene therapy techniques.

Sensitization of Cancer Cells Treated With Cytotoxic Drugs to Fas-Mediated Cytotoxicity

Abstract: Background The transmembrane receptor Fas, together with its protein-binding partner (Fas ligand), is a key regulator of programmed cell death (i.e., apoptosis). Fas and Fas ligand also influence the ability of cytotoxic T lymphocytes and natural killer cells to eliminate tumor cells. However, by inducing apoptosis in activated T cells, the Fas/Fas ligand system may protect some tumor cells from clearance by the immune system. Anticancer drugs enhance Fas ligand expression on the surface of Fas receptor-expressing leukemia cells, thus suggesting that apoptosis caused by these drugs may be mediated via the Fas/Fas ligand system. Purpose This study was conducted to further investigate the relationship between the modulation of Fas receptor gene and protein expression by treatment of cells with cytotoxic drugs and the immune clearance of tumor cells. Methods Fas expression on human HT29 colon carcinoma cells treated with a variety of anticancer drugs (cisplatin, doxorubicin, mitomycin C, fluorouracil, and camptothecin) was analyzed by use of quantitative flow cytometry. Human HCT8R and HCT116 colon carcinoma cells and human U937 leukemia cells were treated with cisplatin only and analyzed in the same way. Fas ligand messenger RNA and protein levels were studied by use of a reverse transcription-polymerase chain reaction assay and by flow cytometry. Fas gene expression and messenger RNA levels in cisplatin-treated HT29 cells were characterized by use of in vitro nuclear run-on and northern blot hybridization assays. The cytotoxic activities of agonistic anti-Fas antibodies, Fas ligand, and allogeneic peripheral blood leukocytes, in the absence or presence of Fas-blocking monoclonal antibodies, against tumor cells were assessed by methylene blue staining and chromium-51 release assays. Results Clinically relevant concentrations of cisplatin, doxorubicin, mitomycin C, fluorouracil, or camptothecin enhanced Fas receptor expression on the plasma membrane of HT29 cells. Cisplatin-mediated increases in Fas expression were confirmed in HCT8R, HCT116, and U937 cells. The enhancement of Fas protein expression was associated with an increased sensitivity of cisplatin-treated tumor cells to agonistic anti-Fas antibodies, to soluble Fas ligand, and to allogeneic peripheral blood leukocyte-mediated cytotoxicity. Each of these effects was blocked by co-treatment of the cells with antagonistic anti-Fas antibody. Conclusion and implications In addition to their direct cytotoxic effects, chemotherapeutic drugs sensitize tumor cells to Fas-mediated cytotoxicity and Fas-dependent immune clearance. On the basis of these findings, new strategies might be developed to improve the efficacy of these drugs.

AP2 inhibits cancer cell growth and activates p21WAF1/CIP1 expression.

Abstract: The 52-kD Activator Protein (AP2) is a DNA-binding transcription factor implicated in signalling terminal differentiation. Profound developmental abnormalities have been recently observed in AP2-null mice. The molecular events by which AP2 promotes differentiation or development are, however, unknown. Increased expression of the universal cell cycle inhibitor p21WAF1/CIP1 occurs in growth-arrested terminally differentiating cells. In a search for cellular factors that could activate p21 during phorbol ester (TPA)-induced differentiation, we identified AP2 as a regulator of p21 expression. Mutagenesis of an AP2 DNA-binding site within a p21 promoter-luciferase reporter inhibited its activation by either AP2 transfection or TPA stimulation. Endogenous p21 protein levels were elevated and DNA synthesis was inhibited in AP2 versus control vector-transfected cells. Overexpression of AP2 in HepG2 human hepatoblastoma and SW480 human colon adenocarcinoma cells inhibited cell division and stable colony formation. These results link the differentiation-associated factor AP2 to negative cell cycle and growth control, possibly through p21 activation.

Enhanced expression of vascular endothelial growth factor in human pancreatic cancer correlates with local disease progression.

Abstract: Vascular endothelial growth factor (VEGF) is an angiogenic polypeptide that has been implicated in cancer growth. In the present study, we characterized VEGF expression in cultured human pancreatic cancer cell lines and determined whether the presence VEGF in human pancreatic cancers is associated with enhanced neovascularization or altered clinicopathological characteristics. VEGF mRNA transcripts were present in all six tested cell lines (ASPC-1, CAPAN-1, MIA-PaCa-2, PANC-1, COLO-357, and T3M4). Immunoblotting with a highly specific anti-VEGF antibody revealed the presence of VEGF protein in all of the cell lines. Northern blot analysis of total RNA revealed a 5.2-fold increase in VEGF mRNA transcript in the cancer samples in comparison with the normal pancreas. Immunohistochemical and in situ hybridization analysis confirmed the expression of VEGF in the cancer cells within the tumor mass. Immunohistochemical analysis of 75 pancreatic cancer tissues revealed the presence of strong VEGF immunoreactivity in the cancer cells in 64% of the cancer tissues. The presence of VEGF in these cells was associated with increased blood vessel number, larger tumor size, and enhanced local spread but not with decreased patient survival. These findings indicate that VEGF is commonly overexpressed in human pancreatic cancers and that this factor may contribute to the angiogenic process and tumor growth in this disorder.

Experimental co-expression of vimentin and keratin intermediate filaments in human breast cancer cells results in phenotypic interconversion and increased invasive behavior

Abstract: The expression of intermediate filament proteins is remarkably tissue specific, which suggests that the intermediate filament type(s) present in cells is somehow related to their biological function. However, in some cancers, particularly malignant breast carcinoma, there is a strong indication that vimentin is co-expressed with keratins, thus presenting as a dedifferentiated or interconverted (between epithelial and mesenchymal) phenotype. In the present study, we recapitulated the interconverted phenotype by developing stable transfectants of MCF-7 human breast cancer cells, termed MoVi clones, to express both vimentin and keratins. Overexpression of vimentin in these cells led to augmentation of motility and invasiveness in vitra. These activities could be transiently down-regulated by vimentin antisense oligonucleotides in MoVi clones and MDA-MB-231 cells (which constitutively co-express keratins and vimentin). Furthermore, in the MoVi experimental transfectants expressing the highest percentage of vimentin-positive cells, their proliferative capacity, clonogenic potential, and tumorigenicity increased. However, the metastatic ability of the MoVi transfectants remained unchanged compared with MCF-7neo controls. The MDA-MB-231 cells metastasized to axillary lymph nodes in a SCID mouse model. Finally, we explored the possibility that potential changes could occur with respect to cell surface integrins. These studies revealed a decrease in the alpha 2- and alpha 3-containing promiscuous integrins, in addition to beta 1 containing integrins, concomitant with an increase in the alpha 6-containing laminin receptor integrin. Further functional analysis of the alpha 6 observation showed an increase in the baptotactic migration of MoVi transfectants toward a laminin substrate. From these data, it is postulated that the ability to co-express vimentin and keratins confers a selective advantage to breast cancer cells in their interpretation of signaling cues from the extracellular matrix; however the addition of vimentin intermediate filaments alone is not sufficient to confer the metastatic phenotype.

Thioredoxin, a gene found overexpressed in human cancer, inhibits apoptosis in vitro and in vivo

Abstract: The redox protein thioredoxin plays an important role in controlling cancer cell growth through regulation of DNA synthesis and transcription factor activity. Thioredoxin is overexpressed by a number of human primary cancers and its expression is decreased during dexamethasone-induced apoptosis of mouse WEHI7.2 thymoma cells. We examined the ability of WEHI7.2 cells stably transfected with human thioredoxin cDNA showing increased levels of cytoplasmic thioredoxin to undergo apoptosis in vitro and in vivo. The cells were protected from apoptosis induced by dexamethasone, staurosporine, etoposide, and thapsigargin, but not by N-acetyl-sphingosine. When inoculated into severe combined immunodeficient mice, the trx-transfected cells formed tumors that showed increased growth compared to wild-type, as well as bcl-2-transfected, WEHI7.2 cells. The trx- and bcl-2-transfected cell tumors both showed less spontaneous apoptosis than tumors formed by the wild-type cells. Unlike tumors formed by the wild-type and bcl-2-transfected WEHI7.2 cells, trx-transfected cell tumors did not show growth inhibition upon treatment with dexamethasone. This study suggests that increased thioredoxin expression in human cancers may result in an increased tumor growth through inhibition of spontaneous apoptosis and a decrease in the sensitivity of the tumor to drug-induced apoptosis.

Aberrant glycolytic metabolism of cancer cells: A remarkable coordination of genetic, transcriptional, post-translational, and mutational events that lead to a critical role for Type II hexokinase

Abstract: For more than two-thirds of this century we have known that one of the most common and profound phenotypes of cancer cells is their propensity to utilize and catabolize glucose at high rates. This common biochemical signature of many cancers, particularly those that are poorly differentiated and proliferate rapidly, has remained until recently a “metabolic enigma.” However, with many advances in the biological sciences having been applied to this problem, cancer cells have begun to reveal their molecular strategies in maintaining an aberrant metabolic behavior. Specifically, studies performed over the past two decades in our laboratory demonstrate that hexokinase, particularly the Type II isoform, plays a critical role in initiating and maintaining the high glucose catabolic rates of rapidly growing tumors. This enzyme converts the incoming glucose to glucose-6-phosphate, the initial phosphorylated intermediate of the glycolytic pathway and an important precursor of many cellular “building blocks.” At the genetic level the tumor cell adapts metabolically by first increasing the gene copy number of Type II hexokinase. The enzyme's gene promoter, in turn, shows a wide promiscuity toward the signal transduction cascades active within tumor cells. It is activated by glucose, insulin, low oxygen “hypoxic” conditions, and phorbol esters, all of which enhance the rate of transcription. Also, the tumor cell uses the tumor suppressor p53, which is usually modified by mutations to debilitate cell cycle controls, to further activate hexokinase gene transcription. This results in both enhanced levels of the enzyme, which binds to mitochondrial porins thus gaining preferential access to mitochondrially generated ATP, and in a decreased susceptibility to product inhibition and proteolytic degradation. Significantly, these multiple strategies all work together to enable tumor cells to develop a metabolic strategy compatible with rapid proliferation and prolonged survival.

Phytoestrogen concentration determines effects on DNA synthesis in human breast cancer cells.

Abstract: Thirteen isoflavonoids, flavonoids, and lignans, including some known phytoestrogens, were evaluated for their effects on DNA synthesis in estrogen-dependent (MCF-7) and -independent (MDA-MB-231) human breast cancer cells. Treatment for 24 hours with most of the compounds at 20-80 microM sharply inhibited DNA synthesis in MDA-MB-231 cells. In MCF-7 cells, on the other hand, biphasic effects were seen. At 0.1-10 microM, coumestrol, genistein, biochanin A, apigenin, luteolin, kaempferol, and enterolactone induced DNA synthesis 150-235% and, at 20-90 microM, inhibited DNA synthesis by 50%. Treatment of MCF-7 cells for 10 days with genistein or coumestrol showed continuous stimulation of DNA synthesis at low concentrations. Time-course experiments with genistein in MCF-7 cells showed effects to be reversed by 48-hour withdrawal of genistein at most concentrations. Induction of DNA synthesis in MCF-7 cells, but not in MDA-MB-231 cells, is consistent with an estrogenic effect of these compounds. Inhibition of estrogen-dependent and -independent breast cancer cells at high concentrations suggests additional mechanisms independent of the estrogen receptor. The current focus on the role of phytoestrogens in cancer prevention must take into account the biphasic effects observed in this study, showing inhibition of DNA synthesis at high concentrations but induction at concentrations close to probable levels in humans.

Inhibition of osteolytic bone metastasis of breast cancer by combined treatment with the bisphosphonate ibandronate and tissue inhibitor of the matrix metalloproteinase-2.

Abstract: Multiple steps are involved in the metastasis of cancer cells from primary sites to distant organs. These steps should be considered in the design of pharmacologic approaches to prevent or inhibit the metastatic process. In the present study, we have compared the effects of inhibiting several steps involved in the bone metastatic process individually with inhibition of both together. The steps we chose were matrix metalloproteinase (MMP) secretion, likely involved in tumor cell invasion, and osteoclastic bone resorption, the final step in the process. We used an experimental model in which inoculation of human estrogen-independent breast cancer MDA-231 cells into the left cardiac ventricle of female nude mice causes osteolytic lesions in bone. To inhibit cancer invasiveness, the tissue inhibitor of the MMP-2 (TIMP-2), which is a natural inhibitor of MMPs, was overexpressed in MDA-231 cells. To inhibit bone resorption, a potent bisphosphonate, ibandronate (4 microg/mouse) was daily administered subcutaneously. Nude mice received either; (a) nontransfected MDA-231 cells; (b) nontransfected MDA231 cells and ibandronate; (c) TIMP-2-transfected MDA-231 cells; or (d) TIMP-2-transfected MDA-231 cells and ibandronate. In mice from group a, radiographs revealed multiple osteolytic lesions. However, in mice from group b or group c, osteolytic lesions were markedly decreased. Of particular note, in animals from group d receiving both ibandronate and TIMP-2-transfected MDA-231 cells, there were no radiologically detectable osteolytic lesions. Survival rate was increased in mice of groups c and d. There was no difference in local enlargement in the mammary fat pad between nontransfected and TIMP-2-transfected MDA-231 cells. These results suggest that inhibition of both MMPs and osteoclastic bone resorption are more efficacious treatment for prevention of osteolytic lesions than either alone, and suggest that when therapies are designed based on the uniqueness of the bone microenvironment and combined with several common steps in the metastatic process, osteolytic bone metastases can be more efficiently and selectively inhibited.

Overexpression of the c-erbB-2 Gene Enhanced Intrinsic Metastasis Potential in Human Breast Cancer Cells without Increasing Their Transformation Abilities

Abstract: Overexpression of the c-erbB-2 gene-encoded p185 has been reported in approximately 30% of human breast cancers and has been correlated with lymph node metastasis and poor prognosis in breast cancer patients. To investigate whether overexpression of p185 can enhance the metastatic potential of human breast cancer cells, we have introduced the human c-erbB-2 gene into the very low p185-expressing MDA-MB-435 human breast cancer cells and established 435.eB transfectants that express higher levels of p185. In this study, we compared the metastatic phenotypes of the parental MDA-MB-435 cells and the 435.eB transfectants. In vivo experimental metastasis assays in which we injected MDA-MB-435 parental cells or 435.eB transfectants into the tail veins of ICR-SCID mice demonstrated that mice injected with p185-overexpressing 435.eB transfectants formed significantly more metastatic tumors than the mice injected with parental and control cells. The changes in experimental metastatic potential in vivo were accompanied by increased invasiveness in vitro. In addition, the secretion of basement membrane-degradative enzymes, which is an important step in the invasion and metastasis process, was also increased in the p185-overexpressing 435.eB transfectants. These results indicated that p185 overexpression can enhance the metastatic potential of MDA-MB-435 human breast cancer cells. To investigate whether enhanced metastatic potential in the p185-overexpressing 435.eB transfectants was the result of increased cancer cell growth and transformation potential, we compared the growth rate, anchorage-independent growth ability, and tumorigenicity of the 435.eB transfectants with that of the parental cells. The transfectants and the parental cells all had similar growth rates and anchorage-independent growth abilities and demonstrated similar tumorigenic potential. These findings suggest that c-erbB-2 is a metastasis-promoting gene for breast cancers that is distinct from other tumor-promoting genes in that the c-erbB-2 gene can enhance the intrinsic metastatic potentials of MDA-MB-435 cells without increasing their transformation abilities.

Targeting gene therapy to cancer: a review.

Abstract: In recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root This is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide This report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment In order to specifically target malignant cells while at the same time sparing normal tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation Although the efficient delivery of DNA to tumor sites remains a formidable task, progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be combined in the future for effective therapy To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC) Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements Another novel possibility that will be discussed is the regulation of therapeutic gene products by tumor-specific gene splicing Gene expression could also be targeted at conditions specific to the tumor microenvironment, such as glucose deprivation and hypoxia We have concentrated on hypoxia-targeted gene expression and this report will discuss our progress in detail Chronic hypoxia occurs in tissue that is more than 100-200 microns away from a functional blood supply In solid tumors hypoxia is widespread both because cancer cells are more prolific than the invading endothelial cells that make up the blood vessels and because the newly formed blood supply is disorganized Measurements of oxygen partial pressure in patients' tumors showed a high percentage of severe hypoxia readings (less than 25 mmHg), readings not seen in normal tissue This is a major problem in the treatment of cancer, because hypoxic cells are resistant to radiotherapy and often to chemotherapy However, severe hypoxia is also a physiological condition specific to tumors, which makes it a potentially exploitable target We have utilized hypoxia response elements (HRE) derived from the oxygen-regulated phosphoglycerate kinase gene to control gene expression in human tumor cells in vitro and in experimental tumors The list of genes that have been considered for use in the treatment of cancer is extensive It includes cytokines and costimulatory cell surface molecules intended to induce an effective systemic immune response against tumor antigens that would not otherwise develop Other inventive strategies include the use of internally expressed antibodies to target oncogenic proteins (intrabodies) and the use of antisense technology (antisense oligonucleotides, antigenes, and ribozymes) This report will concentrate more on novel genes encoding prodrug activating enzymes, so-called suicide genes (Herpes simplex virus thymidine kinase, Escherichia coli nitroreductase, E (ABSTRACT TRUNCATED)

Insulin-like Growth Factor 1 (IGF-1) Alters Drug Sensitivity of HBL100 Human Breast Cancer Cells by Inhibition of Apoptosis Induced by Diverse Anticancer Drugs

Abstract: In this study, we tested the hypothesis that insulin-like growth factor-1 (IGF-1) modulates apoptosis in human breast cancer cells, HBL100, induced by diverse chemotherapeutic drugs. IGF-1 increased cell survival of HBL100 cells treated with 5-fluorouracil (antimetabolite), methotrexate (antimetabolite), tamoxifen (antiestrogen/antiproliferative), or camptothecin (topoisomerase 1 inhibitor) and after serum withdrawal. Elevated cell survival was not due to an increase in cell proliferation by IGF-1, but rather to an inhibition of apoptosis. Evidence for death by apoptosis was supported by cellular morphology and DNA fragmentation. There were no changes observed in Bcl-2 protein or bax mRNA levels. Extracellular matrix (ECM) is known to influence the apoptotic response of cells; therefore, the antiapoptotic effect of IGF-1 on breast cancer cells was examined using different ECMs: laminin, collagen IV, or Matrigel. IGF-1 protected cells from apoptosis induced by methotrexate on all ECMs tested, providing the first evidence that IGF-1 protects against apoptosis in three-dimensional culture systems. These data provide the rationale to search for drugs that lower serum IGF-1 in an effort to improve the efficacy of chemotherapeutic drugs for the treatment of breast cancer.