Showing papers on "Cancer cell published in 1998"

A multidrug resistance transporter from human MCF-7 breast cancer cells

Abstract: MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 663-aa member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.

Gene expression profiles in normal and cancer cells

Abstract: As a step towards understanding the complex differences between normal and cancer cells, gene expression patterns were examined in gastrointestinal tumors. More than 300,000 transcripts derived from at least 45,000 different genes were analyzed. Although extensive similarity was noted between the expression profiles, more than 500 transcripts that were expressed at significantly different levels in normal and neoplastic cells were identified. These data provide insights into the extent of expression differences underlying malignancy and reveal genes that are useful as diagnostic or prognostic markers.

Tumour amplified kinase STK15 / BTAK induces centrosome amplification, aneuploidy and transformation

Abstract: The centrosomes are thought to maintain genomic stability through the establishment of bipolar spindles during cell division, ensuring equal segregation of replicated chromosomes to two daughter cells. Deregulated duplication and distribution of centrosomes have been implicated in chromosome segregation abnormalities, leading to aneuploidy seen in many cancer cell types. Here, we report that STK15 (also known as BTAK and aurora2 ), encoding a centrosome-associated kinase, is amplified and overexpressed in multiple human tumour cell types, and is involved in the induction of centrosome duplication-distribution abnormalities and aneuploidy in mammalian cells. STK15 amplification has been previously detected in breast tumour cell lines 1 and in colon tumours 2 ; here, we report its amplification in approximately 12% of primary breast tumours, as well as in breast, ovarian, colon, prostate, neuroblastoma and cervical cancer cell lines. Additionally, high expression of STK15 mRNA was detected in tumour cell lines without evidence of gene amplification. Ectopic expression of STK15 in mouse NIH 3T3 cells led to the appearance of abnormal centrosome number (amplification) and transformation in vitro . Finally, overexpression of STK15 in near diploid human breast epithelial cells revealed similar centrosome abnormality, as well as induction of aneuploidy. These findings suggest that STK15 is a critical kinase-encoding gene, whose overexpression leads to centrosome amplification, chromosomal instability and transformation in mammalian cells.

CD8+ T Cells Infiltrated within Cancer Cell Nests as a Prognostic Factor in Human Colorectal Cancer

Abstract: The pathophysiological significance of tumor infiltrating lymphocytes remains controversial. To clarify their role, we performed clinicopathological analysis of CD8+ T cells in 131 cases of human colorectal cancer. CD8+ T cells were classified into three groups by their localization: (a) those infiltrated within cancer cell nests; (b) those distributed in the cancer stroma; and (c) those present along the invasive margin (tumor-host interface). Of these, CD8+ T cells within cancer cell nests were most significantly associated with a better survival of patients by both mono- and multivariate analyses. The impact on survival was similar to that of Dukes' staging. Granzyme B+ cytoplasmic granules were detected in lymphocytes within cancer cell nests, confirming their activated, cytotoxic phenotype. CD8 and Ki-67 double immunohistochemistry confirmed higher proliferative activity of CD8+ T cells within cancer cell nests. Our data suggested that human colorectal cancer tissue was infiltrated by various numbers of T cells that had cytotoxic phenotype, contributing to a better survival of patients. This infiltration of colorectal cancer cell nests by CD8+ T cells could be a novel prognostic factor.

Multistep Nature of Metastatic Inefficiency: Dormancy of Solitary Cells after Successful Extravasation and Limited Survival of Early Micrometastases

Abstract: In cancer metastasis, only a small percentage of cells released from a primary tumor successfully form distant lesions, but it is uncertain at which steps in the process cells are lost. Our goal was to determine what proportions of B16F1 melanoma cells injected intraportally to target mouse liver 1) survive and extravasate, 2) form micrometastases (4 to 16 cells) by day 3, 3) develop into macroscopic tumors by day 13, and 4) remain as solitary dormant cells. Using in vivo videomicroscopy, a novel cell accounting assay, and immunohistochemical markers for proliferation (Ki-67) and apoptosis (TUNEL), we found that 1) 80% of injected cells survived in the liver microcirculation and extravasated by day 3, 2) only a small subset of extravasated cells began to grow, with 1 in 40 forming micrometastases by day 3, 3) only a small subset of micrometastases continued to grow, with 1 in 100 progressing to form macroscopic tumors by day 13 (in fact, most micrometastases disappeared), and 4) 36% of injected cells remained by day 13 as solitary cancer cells, most of which were dormant (proliferation, 2%; apoptosis, 3%; in contrast to cells within macroscopic tumors: proliferation, 91%; apoptosis/necrosis, 6%). Thus, in this model, metastatic inefficiency is principally determined by two distinct aspects of cell growth after extravasation: failure of solitary cells to initiate growth and failure of early micrometastases to continue growth into macroscopic tumors.

p53 Activates the CD95 (APO-1/Fas) Gene in Response to DNA Damage by Anticancer Drugs

Abstract: Chemotherapeutic drugs cause DNA damage and kill cancer cells mainly by apoptosis. p53 mediates apoptosis after DNA damage. To explore the pathway of p53-dependent cell death, we investigated if p53-dependent apoptosis after DNA damage is mediated by the CD95 (APO-1/Fas) receptor/ligand system. We investigated hepatoma, gastric cancer, colon cancer, and breast cancer cell lines upon treatment with different anticancer agents known to act via p53 accumulation. Cisplatin, mitomycin, methotrexate, mitoxantrone, doxorubicin, and bleomycin at concentrations present in the sera of patients during therapy led to an upregulation of both CD95 receptor and CD95 ligand. Induction of the CD95 ligand occurred in p53 wild-type (wt), p53 mutant (mt), and p53 deficient (p53−/−) cell lines and at wt and mt conformation of temperature-sensitive p53 mutants. In contrast, upregulation of the CD95 receptor was observed only in cells with wt p53, not in cells with mt or without any p53. Restitution of inducible wt p53 function restored the ability of p53−/− Hep3B cells to upregulate the CD95 receptor in response to anticancer drugs. This rendered the cells sensitive to CD95-mediated apoptosis. In an attempt to understand how CD95 expression is regulated by p53, we identified a p53-responsive element within the first intron of the CD95 gene, as well as three putative elements within the promoter. The intronic element conferred transcriptional activation by p53 and cooperated with p53-responsive elements in the promoter of the CD95 gene. wt p53 bound to and transactivated the CD95 gene, whereas mt p53 failed to induce apoptosis via activation of the CD95 gene. These observations provide a mechanistic explanation for the ability of p53 to contribute to tumor progression and to resistance of cancer cells to chemotherapy.

Ligands for peroxisome proliferator-activated receptorgamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice.

Abstract: Induction of differentiation and apoptosis in cancer cells through ligands of nuclear hormone receptors (NHRs) is a novel and promising approach to cancer therapy. All-trans-retinoic acid (ATRA), an RA receptor-specific NHR ligand, is now used for selective cancers. The NHR, peroxisome proliferator-activated receptor γ (PPARγ) is expressed in breast cancer cells. Activation of PPARγ through a synthetic ligand, troglitazone (TGZ), and other PPARγ-activators cause inhibition of proliferation and lipid accumulation in cultured breast cancer cells. TGZ (10−5 M, 4 days) reversibly inhibits clonal growth of MCF7 breast cancer cells and the combination of TGZ (10−5 M) and ATRA (10−6 M, 4 days) synergistically and irreversibly inhibits growth and induces apoptosis of MCF7 cells, associated with a dramatic decrease of their bcl-2 protein levels. Similar effects are noted with in vitro cultured breast cancer tissues from patients, but not with normal breast epithelial cells. The observed apoptosis mediated by TGZ and ATRA may be related to the striking down-regulation of bcl-2, because forced over-expression of bcl-2 in MCF7 cells cultured with TGZ and ATRA blocks their cell death. TGZ significantly inhibits MCF7 tumor growth in triple immunodeficient mice. Combined administration of TGZ and ATRA causes prominent apoptosis and fibrosis of these tumors without toxic effects on the mice. Taken together, this combination may provide a novel, nontoxic and selective therapy for human breast cancers.

Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression

Abstract: The recently identified PTEN/MMAC1 gene is a candidate tumor suppressor implicated in multiple tumor types based on mutations or homozygous deletions of the gene in certain human cancers. No studies of PTEN/MMAC1 mRNA or protein expression in cancer cells have been reported, primarily because of significant numbers of normal cells contaminating most tumor samples and because of the lack of antibody reagents. We examined PTEN/MMAC1 in advanced prostate cancer for gene mutations or abnormalities in expression by using a series of recently derived xenografts free of normal human cells and a PTEN/MMAC1-specific antibody. Only 1 of 10 tumors contained a homozygous deletion of PTEN/MMAC1, and no mutations were detected in the entire coding region of the remaining nine xenografts. However, five of these showed reduced or absent PTEN/MMAC1 expression by Northern analysis and reverse transcription–PCR of mRNA. PTEN/MMAC1 mRNA expression was restored in nonexpressing prostate cancer cells by in vitro treatment with the demethylating agent 5-azadeoxycytidine. Alterations in PTEN/MMAC1 expression were confirmed at the protein level by immunoblot analysis, and immunohistochemical studies show that the endogenous wild-type PTEN/MMAC1 protein is localized exclusively in the cytoplasm. These results demonstrate that loss of PTEN/MMAC1 expression occurs frequently in advanced prostate cancer.

Ligand for Peroxisome Proliferator-activated Receptor γ (Troglitazone) Has Potent Antitumor Effect against Human Prostate Cancer Both in Vitro and in Vivo

Abstract: Troglitazone, a thiazolidinedione derivative, is a widely used antidiabetic drug that binds and activates peroxisome proliferator-activated receptor gamma (PPARgamma) and enhances insulin sensitivity. It induces differentiation of adipocytes, which highly express PPARgamma. We report that human prostate cancer cells expressed PPARgamma at prominent levels and normal prostate tissues had very low expression. Dose-response clonogenic assays of the PC-3 prostate cancer cell line with troglitazone showed an antiproliferative effect (ED50, 3 x 10(-7) M) and other PPARgamma ligands (BRL49653: ED50, 8 x 10(-8) M; 15-deoxy-delta12,14-prostaglandin J2: ED50, 2 x 10(-6) M; ciglitizone: ED50, not reached; indomethacin: ED50, not reached) showed similar effects. Combinations of troglitazone and a ligand specific for either retinoid X receptor or retinoic acid receptor did not show a synergistic effect. Pulse-exposure to troglitazone (10(-5) M) for different durations showed that 4 days of pulse-exposure to the agent irreversibly inhibited 50% clonal growth of PC-3 cells. Interestingly, PC-3 cells cultured with troglitazone (10(-5) M) showed dramatic morphological changes both by light and electron microscopy, suggesting that the cells became less malignant. Nevertheless, troglitazone did not affect either the cell cycle or several markers of differentiation. LNCaP cells constitutively produced prostate-specific antigen, and levels were markedly enhanced by all-trans-retinoic acid. Troglitazone (10(-5) M, 4 days) decreased by 50% the levels of prostate-specific antigen produced by these cells. In vivo treatment of PC-3 tumors growing in male BNX triple immunodeficient mice with oral troglitazone (500 mg/kg/day) produced significant inhibition of tumor growth (P = 0.01). The only objective side effect of troglitazone in mice was the elevation of serum transaminases. Short-term culture of four surgically obtained human prostate cancer tumors with troglitazone (10(-5) M, 4 days) produced marked and selective necrosis of the cancer cells (about 60%) but not the adjacent normal prostate cells. Taken together, these results suggest that troglitazone may be a useful therapeutic agent for the treatment of prostate cancer, especially in the setting of low disease burden.

Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor.

Abstract: The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal‐transducing Src/p21 ras /Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c‐Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti‐progestins but also by anti‐estrogens. In Cos‐7 cells transfected with the B isoform of progesterone receptor (PR B ), progestin activation of the MAP kinase pathway depends on co‐transfection of ER. A transcriptionally inactive PR B mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PR B does not interact with c‐Src but associates via the N‐terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21 ras /Erk pathway signals received from the agonist‐activated PR B . These findings reveal a hitherto unrecognized cross‐talk between ovarian hormones which could be crucial for their growth‐promoting effects on cancer cells.

Estrogenic Effects of Genistein on the Growth of Estrogen Receptor-positive Human Breast Cancer (MCF-7) Cells in Vitro and in Vivo

Abstract: Genistein, found in soy products, is a phytochemical with several biological activities. In the current study, our research focused on the estrogenic and proliferation-inducing activity of genistein. We have demonstrated that genistein enhanced the proliferation of estrogen-dependent human breast cancer (MCF-7) cells in vitro at concentrations as low as 10 nM, with a concentration of 100 nM achieving proliferative effects similar to those of 1 nM estradiol. Expression of the estrogen-responsive gene pS2 was also induced in MCF-7 cells in response to treatment with a concentration of genistein as low as 1 microM. At higher concentrations (above 20 microM), genistein inhibits MCF-7 cell growth. In vivo, we have shown that dietary treatment with genistein (750 ppm) for 5 days enhanced mammary gland growth in 28-day-old ovariectomized athymic mice, indicating that genistein acts as an estrogen in normal mammary tissue. To evaluate whether the estrogenic effects observed in vitro with MCF-7 cells could be reproduced in vivo, MCF-7 cells were implanted s.c. in ovariectomized athymic mice, and the growth of the estrogen-dependent tumors was measured weekly. Negative control animals received the American Institute of Nutrition (AIN)-93G diet, the positive control group received a new s.c. estradiol (2 mg) pellet plus the AIN-93G diet, and the third group received genistein at 750 ppm in the AIN-93G diet. Tumors were larger in the genistein (750 ppm)-treated group than they were in the negative control group, demonstrating that dietary genistein was able to enhance the growth of MCF-7 cell tumors in vivo. Increased uterine weights were also observed in the genistein-treated groups. In summary, genistein can act as an estrogen agonist in vivo and in vitro, resulting in the proliferation of cultured human breast cancer cells (MCF-7) and the induction of pS2 gene expression. Here we present new information that dietary genistein stimulates mammary gland growth and enhances the growth of MCF-7 cell tumors in ovariectomized athymic mice.

Cell-cell communication in carcinogenesis.

Abstract: To explain the complex carcinogenic process by which a single normal cell in human beings can be converted to an invasive and metastatic cancer cell, a number of experimental findings, epidemiological observations and their associated hypothesis/theories have been integrated in this review. All cancers have been generally viewed as the result of a disruption of the homeostatic regulation of a cell's ability to respond appropriately to extra-cellular signals of the body which trigger intra-cellular signal transducting mechanisms which modulate gap junctional intercellular communication between the cells within a tissue. Normal homeostatic control of these three forms of cell communication determines whether: (a) the cell remains quiescent (Go); (b) enters into the cell proliferation phase; (c) is induced to differentiate; (d) is committed to apoptose; or (e) if it is already differentiated, it can adaptively respond. During the evolution from single cell organisms to multicellular organisms, new cellular/biological functions appeared, namely, the control of cell proliferation ("contact inhibition"), the appearance of the process of differentiation from committed stem cells of the various tissues and the need for programmed cell death or apoptosis. Interestingly, cancer cells have been characterized as cells: (a) having been derived from a stem-like cell; (b) without their ability to control cell growth or without the ability to contact inhibit; (c) which can not terminally differentiate under normal conditions; and (d) having altered ability to apoptosis under normal conditions. During that evolutionary transition from the single cell organism to the multicellular organism, many new genes appeared to accompany these new cellular functions. One of these new genes was the gene coding for a membrane associated protein channel (the gap junction) which between coupled cells, allowed the passive transfer on ions and small molecular weight molecules. A family of over a dozen of these highly evolutionarily-conserved genes (the connexin genes) coded for the connexin proteins. A hexameric unit of these connexins in one cell (a connexon) couples with a corresponding connexon in a contiguous cell to join the cytoplasms. This serves to synchronize either the metabolic or electrotonic functions of cells within a tissue. Most normal cells within solid tissues have functional gap junctional intercellular communication (GJIC) (exceptions are free-standing cells such as red blood cells, neutrophils, and several, if not all, the stem cells). On the other hand, the cancer cells of solid tissues appear to have either dysfunctional homologous or heterologous GJIC. Therefore, among the many differences between a cancer cell and its normal parental cell, the carcinogenic process involves the transition from a normal, GJIC-competent cell to one that is defective in GJIC. The review examines how GJIC can be either transiently or stably modulated by endogenous or exogenesis chemicals or by oncogenes and tumor suppressor genes at the transcriptional, translational, or posttranslational levels. It also uses the gap junction as the biological structure to facilitate cellular/tissue homeostasis to be the integrator for the "stem cell" theory, "disease of differentiation theory", "initiation/promotion/progression" concepts, nature and nurture concept of carcinogenesis, the mutation/ epigenetic theories of carcinogenesis, and the oncogene/ tumor suppressor gene theories of carcinogenesis. From this background, implications to cancer prevention and cancer therapy are generated.

Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells

Abstract: Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2–4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant, N-acetyl-l-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase—programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.

BRCA1 physically associates with p53 and stimulates its transcriptional activity

Abstract: Mutations of the BRCA1 tumor suppressor gene are the most commonly detected alterations in familial breast and ovarian cancer. Although BRCA1 is required for normal mouse development, the molecular basis for its tumor suppressive function remains poorly understood. We show here that BRCA1 increases p53-dependent transcription from the p21WAF1/CIP1 and bax promoters. We also show that BRCA1 and p53 proteins interact both in vitro and in vivo. The interacting regions map, in vitro, to aa 224-500 of BRCA1 and the C-terminal domain of p53. Tumor-derived transactivation-deficient BRCA1 mutants are defective in co-activation of p53-dependent transcription and a truncation mutant of BRCA1 that retains the p53-interacting region acts as a dominant inhibitor of p53-dependent transcription. BRCA1 and p53 cooperatively induce apoptosis of cancer cells. The results indicate that BRCA1 and p53 may coordinately regulate gene expression in their role as tumor suppressors.

Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase

Abstract: The expression of telomerase, the enzyme that synthesizes telomeric DNA de novo, is suppressed in normal somatic human cells but is reactivated during tumorigenesis. This reactivation appears to arrest the normal loss of telomeric DNA incurred as human cells divide. Since continual loss of telomeric DNA is predicted to eventually limit cell proliferation, activation of telomerase in cancer cells may represent an important step in the acquisition of the cell immortalization which occurs during tumor progression. The telomerase holoenzyme is composed of both RNA and protein subunits. In humans, mRNA expression of hTERT (hEST2), the candidate telomerase catalytic subunit gene, appears to parallel the levels of telomerase enzyme activity, suggesting that induction of hTERT is necessary and perhaps sufficient for expression of telomerase activity in tumor cells. To test this model directly, we ectopically expressed an epitope-tagged version of hTERT in telomerase-negative cells and show that telomerase activity was induced to levels comparable to those seen in immortal telomerase-positive cells and that the expressed hTERT protein was physically associated with the cellular telomerase activity. We conclude that synthesis of the hTERT telomerase subunit represents the rate-limiting determinant of telomerase activity in these cells and that this protein, once expressed, becomes part of the functional telomerase holoenzyme.

Tumor cell growth inhibition by caveolin re-expression in human breast cancer cells

Abstract: Cancer development is a multistage process that results from the step-wise acquisition of somatic alterations in diverse genes. Recent studies indicate that caveolin-1 expression correlates with the level of oncogenic transformation in NIH3T3 cells, suggesting that caveolin in caveolae may regulate normal cell proliferation. In order to better understand potential functions of caveolin-1 in cancer development, we have studied expression levels of caveolin-1 in human breast cancer cells, and have found that caveolin expression is significantly reduced in human breast cancer cells compared with their normal mammary epithelial counterparts. When the caveolin cDNA linked to the CMV promoter is transfected into human mammary cancer cells having no detectable endogenous caveolin, overexpression of caveolin-1 resulted in substantial growth inhibition, as seen by the 50% decrease in growth rate and by approximately 15-fold reduction in colony formation in soft agar. In addition, characterization of caveolin-1 expression during cell cycle progression indicates that expression of alpha-caveolin-1 is regulated during cell cycle. Furthermore p53-deficient cells showed a loss in caveolin expression. In summary, the overall expression patterns, its ability to inhibit tumor growth in culture, its regulation during the cell cycle, and the loss of expression in p53-deficient cells all are consistent with an important growth regulating function for caveolin-1 in normal human mammary cells, that needs to be repressed in oncogenic transformation and tumor cell growth.

Expression of TERT in early premalignant lesions and a subset of cells in normal tissues

Abstract: Activation of telomerase, the enzyme that synthesizes the telomere ends of linear chromosomes, has been implicated in human cell immortalization and cancer cell pathogenesis. Enzyme activity is undetectable in most normal cells and tissues, but present in immortal cells and cancer tissues. While expression of TERC, the RNA component of telomerase, is widespread, the restricted expression pattern of TERT, the telomerase catalytic subunit gene, is correlated with telomerase activity, and its ectopic expression in telomerase-negative cells is sufficient to reconstitute telomerase activity and extend cellular lifespan. We have used in situ hybridization to study TERT expression at the single-cell level in normal tissues and in various stages of tumour progression. In normal tissues, including some that are known to be telomerase-negative, TERT mRNA was present in specific subsets of cells thought to have long-term proliferative capacity. This included mitotically inactive breast lobular epithelium in addition to some actively regenerating cells such as the stratum basale of the skin. TERT expression appeared early during tumorigenesis in vivo, beginning with early pre-invasive changes in human breast and colon tissues and increasing gradually during progression, both in the amount of TERT mRNA present within individual cells and in the number of expressing cells within a neoplastic lesion. The physiological expression of TERT within normal epithelial cells that retain proliferative potential and its presence at the earliest stages of tumorigenesis have implications for the regulation of telomerase expression and for the identification of cells that may be targets for malignant transformation.

Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25- hydroxyvitamin D3

Abstract: Epidemiological and laboratory data support a role for vitamin D in the growth and differentiation of human prostatic cells. These findings prompted us to ask whether prostatic cells could convert 25-hydroxyvitamin D3 (25-OH-D3), the major circulating metabolite of vitamin D3, to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active metabolite, in a manner similar to cultured human keratinocytes. Therefore, we investigated three well-characterized human prostate cancer cell lines, LNCaP, DU 145, and PC-3; two primary cultures of cells derived from noncancerous human prostates (one normal and one benign prostatic hyperplasia); and primary cultures of normal human keratinocytes for their ability to synthesize 1,25(OH)2D3. Assays were performed in the presence of 25-OH-D3 as the enzyme substrate and 1,2-dianilinoethane, an antioxidant and free radical scavenger, and in the presence and absence of clotrimazole, a cytochrome P450 inhibitor. DU 145 and PC-3 cells produced 0.31 +/- 0.06 and 0.07 +/- 0.01 pmol of 1,25(OH)2D3/mg protein/h, respectively. No measurable 1,25(OH)2D3 was detected in LNCaP cells. The normal and benign prostatic hyperplasia primary cultures and keratinocyte cultures produced 3.08 +/- 1.56, 1.05 +/- 0.31, and 2.1 +/- 0.1 pmol of 1,25(OH)2D3/mg protein/h, respectively, using a calf thymus receptor binding assay to measure 1,25(OH)2D3 in the presence of 1,2-dianilinoethane. The identity of the analyte as 1,25(OH)2D3 was supported by high performance liquid chromatography using [3H]25-OH-D3 as the enzyme substrate and a solvent system that is specific for 1,25(OH)2D3. The production of 1,25(OH)2D3 in the prostate cancer cell lines and in the primary cultures was completely inhibited in the presence of clotrimazole. This report demonstrates that two of three human prostate cancer cell lines, as well as primary cultures of noncancerous prostatic cells, possess 1alpha-hydroxylase activity and can synthesize 1,25(OH)2D3 from 25-OH-D3. Together with recent data indicating that 1,25(OH)2D3 inhibits the invasiveness of human prostate cancer cells (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997), these data suggest a potential role for 25-OH-D3 in the chemoprevention of invasive prostate cancer.

The cell-surface heparan sulfate proteoglycan glypican-1 regulates growth factor action in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer.

Abstract: Heparan sulfate proteoglycans (HSPGs) play diverse roles in cell recognition, growth, and adhesion. In vitro studies suggest that cell-surface HSPGs act as coreceptors for heparin-binding mitogenic growth factors. Here we show that the glycosylphosphatidylinositol- (GPI-) anchored HSPG glypican-1 is strongly expressed in human pancreatic cancer, both by the cancer cells and the adjacent fibroblasts, whereas expression of glypican-1 is low in the normal pancreas and in chronic pancreatitis. Treatment of two pancreatic cancer cell lines, which express glypican-1, with the enzyme phosphoinositide-specific phospholipase-C (PI-PLC) abrogated their mitogenic responses to two heparin-binding growth factors that are commonly overexpressed in pancreatic cancer: fibroblast growth factor 2 (FGF2) and heparin-binding EGF-like growth factor (HB-EGF). PI-PLC did not alter the response to the non-heparin-binding growth factors EGF and IGF-1. Stable expression of a form of glypican-1 engineered to possess a transmembrane domain instead of a GPI anchor conferred resistance to the inhibitory effects of PI-PLC on growth factor responsiveness. Furthermore, transfection of a glypican-1 antisense construct attenuated glypican-1 protein levels and the mitogenic response to FGF2 and HB-EGF. We propose that glypican-1 plays an essential role in the responses of pancreatic cancer cells to certain mitogenic stimuli, that it is relatively unique in relation to other HSPGs, and that its expression by pancreatic cancer cells may be of importance in the pathobiology of this disorder.

A Novel Three-Pronged Approach to Kill Cancer Cells Selectively: Concomitant Viral, Double Suicide Gene, and Radiotherapy

Abstract: Two obstacles limiting the efficacy of nearly all cancer gene therapy trials are low gene transduction efficiencies and the lack of tumor specificity Recently, a replication-competent, E1B-attenuated adenovirus (ONYX-015) was developed that could overcome these limitations, because it was capable of efficiently and selectively destroying tumor cells lacking functional p53 In an attempt to improve both the efficacy and safety of this approach, we constructed a similar adenovirus (FGR) containing a cytosine deaminase (CD)/herpes simplex virus type-1 thymidine kinase (HSV-1 TK) fusion gene, thereby allowing for the utilization of double-suicide gene therapy, which has previously been demonstrated to produce significant antitumor effects and potentiate the therapeutic effects of radiation The FGR virus exhibited the same tumor cell specificity and replication kinetics as the ONYX-015 virus in vitro Importantly, both the CD/5-FC and HSV-1 TK/GCV suicide gene systems markedly enhanced the tumor cell-specific cytopathic effect of the virus, and, as expected, sensitized tumor cells to radiation By contrast, neither the FGR virus nor either suicide gene system showed significant toxicity to normal human cells Both suicide gene systems could be used to suppress viral replication effectively, thereby providing a means to control viral spread The results support the thesis that the three-pronged approach of viral therapy, suicide gene therapy, and radiotherapy may represent a powerful and safe means of selectively destroying tumor cells in vivo

Predictive value of vascular endothelial growth factor (VEGF) in metastasis and prognosis of human colorectal cancer

Abstract: Vascular endothelial growth factor (VEGF) may affect the phenotype of cancer cells, such as growth velocity and metastatic potential, due to its probable multifunctional property including a mitogenic activity for vascular endothelial cells. The present study was designed to investigate the association of VEGF mRNA expression with progression and metastasis of human colorectal cancer. The level of VEGF mRNA expression was quantified by Northern blot hybridization in tumorous and non-tumorous tissues obtained from 60 primary colorectal cancer patients. The ratio of the former to the latter was defined as the VEGF T/N ratio, and the prognostic significance of this ratio, following surgery, in addition to the relationship to progression and metastatic potential, was evaluated. The value of the VEGF T/N ratio was significantly correlated with the depth of tumour infiltration (P=0.046), the incidence of liver metastasis (P < 0.0001) and lymph node metastasis (P=0.036). Patient prognosis was estimated by the Kaplan-Meier method and the log-rank test. When the VEGF T/N ratio was higher than 4.8 for which the chi2 value of the log-rank test was maximal, the tumour was defined as showing overexpression of VEGF mRNA. Patients with overexpression of VEGF mRNA demonstrated poorer survival than patients without overexpression of VEGF mRNA (P < 0.001). The overall estimated hazard ratio for death in patients with overexpression of VEGF mRNA was 1.94 according to a multivariate analysis (P=0.005). Thus, VEGF is associated with the progression, invasion and metastasis of colorectal cancer, and overexpression of VEGF mRNA in the primary tumour is assumed to be closely correlated with poor prognosis in colorectal cancer patients. Moreover, the VEGF T/N ratio may be used as an independent prognostic marker in colorectal cancer patients.

Tumor Cell-associated Hyaluronan as an Unfavorable Prognostic Factor in Colorectal Cancer

Abstract: Hyaluronan (HA) is a linear high molecular weight extracellular polysaccharide. It is thought to be involved in mitosis and the enhancement of wound healing, tumor invasion, and metastasis. Because its clinical relevance in cancer has not been explored, we scored HA in colorectal adenocarcinoma and studied its relationship with patient survival. A specific probe prepared from cartilage proteoglycan aggregates was used to stain paraffin-embedded tumor samples from 202 colorectal adenocarcinoma patients treated in Kuopio University Hospital and followed up for a mean of 14 years. The hypothesis that the percentage of HA-positive carcinoma cells (HA%) and HA intensity in cancer cells correlated with survival was tested with the log-rank test, hazard ratios, and their confidence intervals. Ninety-three % of tumors had at least a proportion of carcinoma cells positive for HA. HA intensity in tumor epithelium was stronger in Dukes' stages C and D tumors and in high-grade tumors. The cancer-related survival rate was lower among patients with strong HA intensity in tumor epithelium (P < 0.001) and high HA% (P < 0.001). Recurrence-free survival was also shorter in patients with an intense signal for HA (P = 0.001) and high HA% in tumor epithelium (P = 0.04). HA intensity in tumor epithelium independently predicted survival and recurrence-free survival (Cox's analysis). We conclude that a high proportion of HA-positive cancer cells and high intensity of the HA-signal predicts a poor survival rate. The abnormal expression of HA in the neoplastic colon epithelial cells is suggested to provide a distinct advantage for invasive growth and metastasis.

Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy.

Abstract: Heat shock proteins (Hsps) are induced in vitro by several cytotoxic drugs; in human breast cancer cells these proteins appear to be involved in anti-cancer drug resistance. The present report was designed to analyze whether chemotherapy affects in vivo the expression of Hsp27, Hsp70, Hsc70 and Hsp90 in breast cancer patients treated with induction chemotherapy and whether these proteins may be determinants of tumor resistance to drug administration. We have analyzed 35 biopsies from breast cancer patients treated with induction chemotherapy. Expression of the Hsps in the tumors was compared with (i) histological and clinical responses to chemotherapy, (ii) tumor cell proliferation measured by proliferating cell nuclear antigen (PCNA) immunostaining and nucleolar organizer regions (AgNORs) staining and (iii) the expression of estrogen and progesterone receptors. We also compared disease-free survival (DFS) and overall survival (OS) with the expression of the Hsps studied. After chemotherapy, nuclear Hsp27 and Hsp70 expression was increased and Hsp70 and Hsc70 cytoplasmic expression was decreased. A high nuclear proportion of Hsp70 in tumor cells (>10%) correlated significantly with drug resistance. We also observed that patients whose tumors expressed nuclear or a high cytoplasmic proportion (>66%) of Hsp27 had shorter DFS. The combination of Hsp27 and Hsp70 levels showed a strong correlation with DFS. Neither the cellular proliferation nor the levels of steroid receptors showed any significant difference before or after drug administration or during follow-up of patients. Our results suggest that Hsp27 and Hsp70 are involved in drug resistance in breast cancer patients treated with combination chemotherapies.

p53 and vascular endothelial growth factor regulate tumor growth of NOS2-expressing human carcinoma cells

Abstract: The finding of frequent nitric oxide synthase expression in human cancers indicates that nitric oxide has a pathophysiological role in carcinogenesis. To determine the role of nitric oxide in tumor progression, we generated human carcinoma cell lines that produced nitric oxide constitutively. Cancer cells expressing inducible nitric oxide synthase that had wild-type p53 had reduced tumor growth in athymic nude mice, whereas those with mutated p53 had accelerated tumor growth associated with increased vascular endothelial growth factor expression and neovascularization. Our data indicate that tumor-associated nitric oxide production may promote cancer progression by providing a selective growth advantage to tumor cells with mutant p53, and that inhibitors of inducible nitric oxide synthase may have therapeutic activity in these tumors.

Identification of a meiosis-specific protein as a member of the class of cancer/testis antigens

Abstract: Little is known about the function of human cancer/testis antigens (CTAs), such as MAGE, BAGE, GAGE, HOM-MEL-40, and NY-ESO-1, the expression of which is restricted to human malignancies and testis. When screening a cDNA expression library enriched for testis-specific representative long transcripts for reactivity with high-titered IgG antibodies from the serum of a patient with renal cell carcinoma, one repeatedly detected antigen, designated HOM-TES-14, turned out to be encoded by the synaptonemal complex protein 1 (SCP-1) gene. SCP-1 is known to be selectively expressed during the meiotic prophase of spermatocytes and is involved in the pairing of homologous chromosomes, an essential step for the generation of haploid cells in meiosis I. Investigation of a broad spectrum of normal and malignant tissues revealed expression of SCP-1 transcripts and antigen selectively in a variety of neoplastic tissues and tumor cell lines. Immunofluorescence microscopy analysis with specific antiserum showed a cell cycle phase-independent nuclear expression of SCP-1 protein in cancer cells. SCP-1 differs from other members of the class of CTA by its localization on chromosome 1 and its frequent expression in malignant gliomas, breast, renal cell, and ovarian cancer. The aberrant expression of SCP-1 in tumors might contribute to their genomic instability and suggests that the functional role of other CTA might also relate to meiosis.

Establishing human prostate cancer cell xenografts in bone: Induction of osteoblastic reaction by prostate‐specific antigen‐producing tumors in athymic and SCID/bg mice using LNCaP and lineage‐derived metastatic sublines

Abstract: LNCaP lineage-derived human prostate cancer cell lines C4-2 and C4-2B4 acquire androgen independence and osseous metastatic potential in vivo. Using C4-2 and C4-2B4 the goals of the current investigation were 1) to establish an ideal bone xenograft model for prostate cancer cells in intact athymic or SCID/bg mice using an intraosseous route of tumor cell administration and 2) to compare prostate cancer metastasis by administering cells either through intravenous (i.v.) or intracardiac administration in athymic or SCID/bg mice. Subsequent to tumor cell administration, prostate cancer growth in the skeleton was assessed by radiographic bone density, serum prostate-specific antigen (PSA) levels, presence of hematogenous prostate cancer cells and histopathologic evaluation of tumor specimens in the lymph node and skeleton. Our results show that whereas LNCaP cells injected intracardially failed to develop metastasis, C4-2 cells injected similarly had the highest metastatic capability in SCID/bg mice. Retroperitoneal and mediastinal lymph node metastases were noted in 3/7 animals, whereas 2/7 animals developed osteoblastic spine metastases. Intracardiac injection of C4-2 in athymic hosts produced spinal metastases in 1/5 animals at 8-12 weeks post-injection; PC-3 injected intracardially also metastasized to the bone but yielded osteolytic responses. Intravenous injection of either LNCaP or C4-2 failed to establish tumor colonies. Intrailiac injection of C4-2 but not LNCaP nor C4-2B4 cells in athymic mice established rapidly growing tumors in 4/8 animals at 2-7 weeks after inoculation. Intrafemoral injection of C4-2 (9/16) and C4-2B4 (5/18) but not LNCaP (0/13) cells resulted in the development of osteoblastic bone lesions in athymic mice (mean: 6 weeks, range: 3-12 weeks). In SCID/bg mice, intrafemoral injection of LNCaP (6/8), C4-2 (8/8) and C4-2B4 (8/8) cells formed PSA-producing, osteoblastic tumors in the bone marrow space within 3-5 weeks after tumor cell inoculation. A stepwise increase of serum PSA was detected in all animals. Reverse transcription-polymerase chain reaction (RT-PCR) to detect hematogenously disseminated prostate cancer cells could not be correlated to either serum PSA level or histological evidence of tumor cells in the marrow space. We have thus established a PSA-producing and osteoblastic human prostate cancer xenograft model in mice.