Showing papers on "Cancer cell published in 1994"

Cell cycle control and cancer

Abstract: Multiple genetic changes occur during the evolution of normal cells into cancer cells. This evolution is facilitated in cancer cells by loss of fidelity in the processes that replicate, repair, and segregate the genome. Recent advances in our understanding of the cell cycle reveal how fidelity is normally achieved by the coordinated activity of cyclin-dependent kinases, checkpoint controls, and repair pathways and how this fidelity can be abrogated by specific genetic changes. These insights suggest molecular mechanisms for cellular transformation and may help to identify potential targets for improved cancer therapies.

Derivation of androgen-independent human LNCaP prostatic cancer cell sublines: Role of bone stromal cells

Abstract: A model of human prostate cancer was established to study cellular interaction between prostate cancer and bone stroma in vivo. In this model, subcutaneous co-injection of 2 non-tumorigenic human cell lines-LNCaP, a prostate cancer cell line, and MS, a bone stromal cell line-into intact adult male mice resulted in formation of carcinomas that secreted prostate-specific antigen (PSA), a clinically useful human serum prostate cancer marker. In castrated hosts, upon cellular interaction with bone fibroblasts, we observed the progression of these tumors from an androgen-dependent (AD) to an androgen-independent state (AI). We derived 4 LNCaP cell sublines from the chimeric LNCaP/MS tumors: the M subline from intact hosts and the C4, C4-2 and C5 sublines from castrated hosts. The LNCaP sublines had chromosomal markers similar to those of the parental LNCaP cells and distinctly different from those of the MS bone stromal cell line. Although the parental and derived cell lines expressed similar steady-state levels of orni-thine decarboxylase transcript, the sublines expressed 5- to 10-fold higher basal steady-state levels of PSA transcript than did the parental LNCaP cell line. The LNCaP sublines formed 13- to 26-fold more soft-agar colonies than the parental LNCaP cell line. The sublines became tumorigenic, yielding an incidence of tumors in intact athymic mice of 7-75%. The LNCaP sublines C4 and C5 (but not the parental and M cell line) formed tumors in castrated hosts when co-injected with bone fibroblasts. A second-generation LNCaP subline, C4-2, was derived from a chimeric tumor induced by co-inoculating castrated mouse with C4 cells and MS cells. We found that C4-2 subline was tumorigenic when inoculated into castrated hosts in the absence of inductive fibroblasts. Moreover, C4-2 was the only subline capable of forming soft-agar colonies when cultured in serum-free medium. In comparison with the parental LNCaP cells, the C4-2 subline expressed lower steady-state levels of androgen receptor (AR) protein and mRNA transcript and lost its androgen responsiveness in vitro. Our results suggest that certain genetic traits of prostate cancer cells may be selected or altered through an “adaptive” mechanism that involves cellular interaction with the bone stromal cells. © 1994 Wiley-Liss, Inc.

Antibody to HER-2/neu receptor blocks DNA repair after cisplatin in human breast and ovarian cancer cells.

Abstract: Approximately 30% of human breast and ovarian cancers have amplification and/or overexpression of HER-2/neu gene which encodes a cell surface growth-factor receptor. Overexpression of this receptor, p185HER-2/neu, is associated with poor outcome and may predict clinical response to chemotherapy. Antibodies to HER-2/neu receptor have a cytostatic effect in suppressing growth of cells with overexpression of p185HER-2/neu. To elicit a cytocidal effect, therapy with antireceptor antibody was used in combination with the DNA-damaging drug, cisplatin, and this combined treatment produced a synergistic decrease in cell growth. In addition, antibody mediated an increased sensitivity to cisplatin in drug-resistant ovarian carcinoma cells containing multiple copies of HER-2/neu gene. To evaluate the mechanism for this synergy, unscheduled DNA synthesis was measured in cancer cells using incorporation of [3H]thymidine and autoradiography, and formation and repair of cisplatin-induced DNA adducts was also measured. Treatment with cisplatin led to a marked, dose-dependent increase in unscheduled DNA synthesis which was significantly reduced by combined treatment with antireceptor antibody in HER-2/neu-overexpressing cells. Therapy with antibody to HER-2/neu receptor also led to a 35-40% reduction in repair of cisplatin-DNA adducts after cisplatin exposure and, as a result, promoted drug-induced killing in target cells. This phenomenon which we term receptor-enhanced chemosensitivity may provide a rationale for more selective targeting and exploitation of overexpressed growth factor receptors in cancer cells, thus leading to new strategies for clinical intervention.

Cyclin D1 induction in breast cancer cells shortens G1 and is sufficient for cells arrested in G1 to complete the cell cycle

Abstract: The sequential transcriptional activation of cyclins, the regulatory subunits of cell-cycle-specific kinases, is thought to regulate progress through the cell cycle. Cyclins are therefore potential oncogenes, and cyclin D1 overexpression and/or amplification at its genomic locus, 11q13, are common features of several human cancers. Induction of cyclin D1 is an early response to mitogenic stimulation in several cell types, but the consequences of altered expression of this gene in human cells of epithelial origin remain undefined. We assessed the effects of alterations of cyclin D1 expression in human breast cancer cells by generating T-47D cells expressing human cyclin D1 under the control of a zinc-responsive metallothionein promoter. In cycling cells induction of cyclin D1 after zinc treatment resulted in an increase in the number of cells progressing through G1 and in the rate of transition from G1 to S phase, indicating that cyclin D1 is rate-limiting for progress through G1 phase. In cells arrested in early G1 phase after growth factor deprivation, zinc induction of cyclin D1 was sufficient for completion of the cell cycle, a process requiring growth factor stimulation in control cells. These data demonstrate a critical role for cyclin D1 in human breast cancer cell-cycle control and suggest that deregulated expression of cyclin D1 is likely to reduce dependence on normal physiological growth stimuli, thereby providing a growth advantage to tumor cells and a potential mechanism of resistance to endocrine therapy.

Creatine kinase in non-muscle tissues and cells

Abstract: Over the past years, a concept for creatine kinase function, the ‘PCr-circuit’ model, has evolved. Based on this concept, multiple functions for the CK/PCr-system have been proposed, such as an energy buffering function, regulatory functions, as well as an energy transport function, mostly based on studies with muscle. While the temporal energy buffering and metabolic regulatory roles of CK are widely accepted, the spatial buffering or energy transport function, that is, the shuttling of PCr and Cr between sites of energy utilization and energy demand, is still being debated. There is, however, much circumstantial evidence, that supports the latter role of CK including the distinct, isoenzyme-specific subcellular localization of CK isoenzymes, the isolation and characterization of functionally coupledin vitro microcompartments of CK with a variety of cellular ATPases, and the observed functional coupling of mitochondrial oxidative phosphorylation with mitochondrial CK. New insight concerning the functions of the CK/PCr-system has been gained from recent M-CK null-mutant transgenic mice and by the investigation of CK localization and function in certain highly specialized non-muscle tissues and cells, such as electrocytes, retina photoreceptor cells, brain cells, kidney, salt glands, myometrium, placenta, pancreas, thymus, thyroid, intestinal brush-border epithelial cells, endothelial cells, cartilage and bone cells, macrophages, blood platelets, tumor and cancer cells. Studies with electric organ, includingin vivo31P-NMR, clearly reveal the buffer function of the CK/PCr-system in electrocytes and additionally corroborate a direct functional coupling of membrane-bound CK to the Na+/K+-ATPase. On the other hand, experiments with live sperm and recentin vivo31P-NMR measurements on brain provide convincing evidence for the transport function of the CK/PCr-system. We report on new findings concerning the isoenzyme-specific cellular localization and subcellular compartmentation of CK isoenzymes in photoreceptor cells, in glial and neuronal cells of the cerebellum and in spermatozoa. Finally, the regulation of CK expression by hormones is discussed, and new developments concerning a connection of CK with malignancy and cancer are illuminated. Most interesting in this respect is the observed upregulation of CK expression by adenoviral oncogenes.

PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth.

Abstract: PRL-1 is a particularly interesting immediate-early gene because it is induced in mitogen-stimulated cells and regenerating liver but is constitutively expressed in insulin-treated rat H35 hepatoma cells, which otherwise show normal regulation of immediate-early genes. PRL-1 is expressed throughout the course of hepatic regeneration, and its expression is elevated in a number of tumor cell lines. Sequence analysis reveals that PRL-1 encodes a 20-kDa protein with an eight-amino-acid consensus protein tyrosine phosphatase (PTPase) active site. PRL-1 is able to dephosphorylate phosphotyrosine substrates, and mutation of the active-site cysteine residue abolishes this activity. As PRL-1 has no homology to other PTPases outside the active site, it is a new type of PTPase. PRL-1 is located primarily in the cell nucleus. Stably transfected cells which overexpress PRL-1 demonstrate altered cellular growth and morphology and a transformed phenotype. It appears that PRL-1 is important in normal cellular growth control and could contribute to the tumorigenicity of some cancer cells.

The gamma 2 chain of kalinin/laminin 5 is preferentially expressed in invading malignant cells in human cancers.

Abstract: All known laminin isoforms are cross-shaped heterotrimeric molecules, consisting of one heavy alpha chain and two light beta and gamma chains. Recently, a cDNA encoding a new gamma chain from laminin 5 (also known as kalinin) was sequenced. This chain, named gamma 2, showed extended homology to the classical gamma 1 chain but differed from this by lacking the terminal globular domain. Recent data, indicating an important role of the gamma 2 chain gene in establishing adhesion contacts between epithelial cells and basement membranes, prompted us to investigate whether the gamma 2 chain gene is aberrantly expressed in cancer tissue, and if so whether its localization could provide clues to its possible role in cancer dissemination. Routinely processed tissue specimens from 36 cases of human cancer were investigated, including 16 cases of colon adenocarcinoma, 7 ductal mammary carcinomas, 4 squamous cell carcinomas, 3 malignant melanomas and 6 sarcomas. In situ hybridization for the detection of mRNAs for the gamma 2 chain and for the classical laminin chains alpha 1, beta 1, and gamma 1 was performed using S-35 labeled antisense RNA probes. As positive control of the specificity of the gamma 2 chain mRNA detection, two different anti-sense probes derived from two nonoverlapping cDNA clones were used. Malignant cells were found to express the gamma 2 chain in 29 of the 30 carcinomas studied and the expression was particularly high in cancer cells located at the invasion front. In contrast, mesenchymally derived cancer cells in three different types of sarcomas did not express the gamma 2 chain. In colon cancer there was a clear histological correlation between the expression of gamma 2 chain by cancer cells and their engagement in tumor budding processes. Laminin chains alpha 1, beta 1, and gamma 1 were weakly expressed throughout cancerous areas with no apparent correlation to sites of invasion. The aberrant expression of the gamma 2 chain gene seen in invasively growing cancer cells point to a role of this molecule in establishing focal adhesions of cancer cells to the extracellular matrix during their migration through surrounding normal tissue.

A Truncated β-Catenin Disrupts the Interaction between E-Cadherin and α-Catenin: A Cause of Loss of Intercellular Adhesiveness in Human Cancer Cell Lines

Abstract: Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and are considered to work as an invasion suppressor system of cancer cells. They form a molecular complex with catenins, a group of cytoplasmic proteins including alpha- and beta-catenins. While alpha-catenin has been demonstrated to be crucial for cadherin function, the role of beta-catenin is not yet fully understood. In this study, we analyzed the cadherin-catenin system in two human cell lines, HSC-39 and its putative subline HSC-40A, derived from a signet ring cell carcinoma of stomach. These cells grow as loose aggregates or single cells, suggesting that their cadherin system is not functional. In these cell lines, an identical 321-base pair in-frame mRNA deletion of beta-catenin was identified; this led to a 107-amino-acid deletion in the NH2-terminal region of the protein. Southern blot analysis disclosed a homozygous deletion in part of the beta-catenin gene. On the other hand, these cells expressed E-cadherin, alpha-catenin, and plakoglobin of normal size. Immunoprecipitation analyses showed that E-cadherin was coprecipitated with the mutated beta-catenin but not with alpha-catenin, and antibodies against beta-catenin did not copurify alpha-catenin. However, the recombinant fusion protein containing wild-type beta-catenin precipitated alpha-catenin from these cells. These results suggest that the dysfunction of E-cadherin in these cell lines is due primarily to its failure to interact with alpha-catenin, and that this defect results from the mutation in beta-catenin. Thus, it is most likely that the association between E-cadherin and alpha-catenin is mediated by beta-catenin, and that this process is blocked by NH2-terminal deletion in beta-catenin. These findings indicate that genetic abnormality of beta-catenin is one of the mechanisms responsible for loosening of cell-cell contact, and may be involved in enhancement of tumor invasion in human cancers.

Quercetin potentiates the effect of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: P-glycoprotein as a possible target.

Abstract: This study demonstrates that the flavonoid quercetin (Q), a plant-derived compound with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of Adriamycin (ADR) on MCF-7 ADR-resistant human breast cancer cells. The effect of Q was dose-dependent at concentrations ranging between 1 and 10 μM. Since ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of Q and related flavonoids of Pgp activity in cytofluorographic efflux experiments with the fluorescent dye rhodamine 123 (Rh 123). Our results indicate that Q and 3-OMe Q (3′,4′,7-trimethoxyquercetin) but not the 3-rhamnosylglucoside of Q (rutin) inhibit the Pgp pump-efflux activity in a dose-related manner. Moreover, 10 μM Q reduces the expression of the immunoreactive Pgp in MCF-7 ADR-resistant cells as evaluated by cytofluorimetric assay. In conclusion, these findings provide a further biological basis for the potential therapeutic application of Q as an anticancer drug either alone or in combination with ADR in multidrug-resistant breast tumor cells.

Growth Suppression of Human Head and Neck Cancer Cells by the Introduction of a Wild-Type p53 Gene via a Recombinant Adenovirus

Abstract: Mutations of the p53 gene constitute one of the most frequent genetic alterations in squamous cell carcinoma of the head and neck (SCCHN). In this study, we introduced wild-type p53 into two separate SCCHN cell lines via a recombinant adenoviral vector, Ad5CMV-p53. Northern blotting showed that following infection by the wild-type p53 adenovirus (Ad5CMV-p53), cells produced up to 10-fold higher levels of exogenous p53 mRNA than cells treated with vector only (without p53). Western blotting showed that the increased levels of p53 protein produced in the Ad5CMV-p53-infected cells were a reflection of p53 mRNA expression. In vitro growth assays revealed growth arrest following Ad5CMV-p53 infection as well as cell morphological changes consistent with apoptosis. In vivo studies in nude mice with established s.c. squamous carcinoma nodules showed that tumor volumes were significantly reduced in mice that received peritumoral infiltration of Ad5CMV-p53. These data suggest that Ad5CMV-p53 may be further developed as a potential novel therapeutic agent for SCCHN since introduction of wild-type p53 into SCCHN cell lines attenuates their replication and tumor growth.

Expression of epidermal growth factor receptor in bladder cancer as related to established prognostic factors, oncoprotein (c-erbB-2, p53) expression and long-term prognosis.

Abstract: The expression of epidermal growth factor receptor (EGFR) was studied immunohistochemically in 234 cases of transitional cell bladder cancer. EGFR was overexpressed in 35% of cases and distinct nuclear localisation of EGFR positivity was found in 31% of the tumours. Overexpression was related to invasive growth, grade 2-3 histology, non-papillary type, DNA aneuploidy and high proliferation rate of cancer cells. The expressions of p53 and EGFR were interrelated, while expression of c-erbB-2 was independent of EGFR expression. Progression of superficial tumours, recurrence-free survival and survival were independently related to overexpression of EGFR in multivariate analysis. T category, S-phase fraction and non-papillary type included all the available prognostic information when the entire cohort was analysed by multivariate methods. The results show that overexpression of EGFR is related to several malignant features and prognosis in superficial bladder cancer. Moreover, the results suggest that overexpression of EGFR is usually a late event in bladder cancer development related to genetic instability rather than an early event in malignant transformation. Further studies are still needed to establish whether the direct measurement of cell proliferation or analysis of growth factor receptors and other oncoproteins gives more accurate prognostic information in bladder cancer.

Osteolytic bone metastasis in breast cancer

Abstract: Metastasis of breast cancer cells to bone consists of multiple sequential steps. To accomplish the process of metastasis to bone, breast cancer cells are required to intrinsically possess or acquire the capacities that are necessary for them to proliferate, invade, migrate, survive, and ultimately arrest in bone. These capacities are essential for any cancer cells to develop distant metastases in organs such as lungs and liver as well as bone. Once breast cancer cells arrest in bone, bone is a storehouse of a variety of cytokines and growth factors and thus provides an extremely fertile environment for the cells to grow. However, breast cancer cells are unable to progress in bone unless they destroy bone with the assistance of bone-resorbing osteoclasts. Thus, the capacity of breast cancer cells to collaborate with osteoclasts is likely to be specific and is likely critical for them to cause osteolytic bone metastases. Evidence to support the concept that there is an intimate relationship between breast cancer cells and osteoclasts is described using an in vivo bone metastasis model in which human breast cancer cells are inoculated into the left ventricle of nude mice. The roles of cell adhesion molecules including cadherins and laminin and matrix metalloproteinases in the development of osteolytic bone metastases by breast cancer are also discussed.

Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells

Abstract: Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.

Coexpression of the c-met Proto-oncogene and Hepatocyte Growth Factor in Human Pancreatic Cancer

Abstract: The c-met proto-oncogene encodes a transmembrane tyrosine kinase receptor (MET) that has the capacity to modulate cell proliferation and differentiation; it is activated by the hepatocyte growth factor Using a highly specific anti-MET antibody we found mild MET immunoreactivity in acinar, ductal, and islet cells in the normal human pancreas and intense MET immunoreactivity in many of the duct-like cancer cells in 14 of 16 human pancreatic adenocarcinomas Intense MET immunoreactivity was also evident in the ductal cells in regions adjacent to the cancer cells Northern blot analysis of total RNA revealed that, by comparison with the normal pancreas, pancreatic cancers exhibited a 7-fold (P < 001) increase in c-met mRNA levels Hepatocyte growth factor mRNA levels were increased 10-fold (P < 005) in the same cancers The concomitant over-expression of c-met and hepatocyte growth factor in human pancreatic cancers suggests that there is excessive activation of c-met-dependent signaling pathways that may contribute to pancreatic cancer cell growth in vivo

p53 and human cancers.

Abstract: Mutations in the p53 gene are one of the commonest specific genetic changes found in human cancer. The p53 gene is not required for normal development but lack of p53 function confers an enormously elevated risk of developing cancer, thus it seems truly to act as a tumour suppressor gene. The p53 protein is normally present in minute amounts in cells but when cells are exposed to genotoxic stimuli p53 levels rise rapidly and initiate a programme of cell death, probably by means of transcriptional regulation. This response is lost in many tumour cells as they have either inactivated their p53 genes by mutation or blocked the activity of p53 through the production of proteins that bind to it and neutralise it. Mutant p53 proteins accumulate to high levels in many cancer cells and the p53 protein and the p53 response to DNA damage represent key points for therapeutic intervention.

Epidermal Growth Factor-mediated Apoptosis of MDA-MB-468 Human Breast Cancer Cells

Abstract: MDA-MB-468 human breast cancer cells lack estrogen receptors, overexpress epidermal growth factor (EGF) receptors, and are growth inhibited by EGF. We show that treatment of MDA-MB-468 cells with EGF leads to inhibition of cell proliferation, fragmentation of DNA into nucleosomal oligomers, and the development of apoptotic morphology. This treatment is associated with increased expression of c-myc, c-fos, jun family members, and transforming growth factor beta 1 mRNA and with partial proteolytic cleavage of poly(ADP-ribose) polymerase and lamin B. The observation that EGF can mediate apoptosis in EGF receptor-overexpressing cells has important implications for clinical efforts directed at the EGF receptor.

Modulation of estrogen receptor mRNA expression by melatonin in MCF-7 human breast cancer cells.

Abstract: Melatonin, the hormonal product of the pineal gland, has been shown to inhibit the development of mammary tumors in vivo and the proliferation of MCF-7 human breast cancer cells in vitro by mechanisms not yet identified. However, previous studies have demonstrated that melatonin significantly decreased estrogen-binding activity and the expression of immunoreactive estrogen receptor (ER) in MCF-7 breast cancer cells. To determine the mechanism(s) by which melatonin regulates ER expression in MCF-7 cells, the relationship between the level of steady state ER mRNA and the rate of ER gene transcription were examined in response to melatonin. Physiological concentrations of melatonin decreased steady state levels of ER mRNA expression in a dose- and time-specific manner. This decrease was not dependent upon the presence of estrogen since similar decreases in steady state ER mRNA levels were seen in MCF-7 cells cultured in both complete and estrogen-depleted media. The decreased expression of ER mRNA in respons...

Overexpression of metalloproteinase inhibitor in B16F10 cells does not affect extravasation but reduces tumor growth.

Abstract: It is widely accepted that a major role of matrix metalloproteinases in the metastatic process is degradation of basement membrane during cancer cell invasion. We tested the hypothesis that the reduction in metastatic potential which has been demonstrated for B16F10 melanoma cells genetically engineered to overexpress tissue inhibitor of metalloproteinase-1 (TIMP-1) is caused by a decrease in their ability to extravasate. Using intravital videomicroscopy of chick embryo chorioallantoic membrane, we studied extravasation of B16F10 cells and B16F10 cells transfected to overexpress TIMP-1. More than 800 cells in 36 chick embryos were analyzed for each cell line during 72 h postinjection. TIMP-1 upregulation had no effect on the time course of extravasation, virtually all cells from both cell lines having extravasated by 36 h. We also studied the morphology of micrometastases at days 3 and 7. Lack of contact between cancer cells within micrometastases at day 3 and reduction in size and number of tumors at day 7 were observed for TIMP-1 overexpressor cells compared to B16F10. Our findings illustrate that the imbalance between TIMP and metalloproteinases created by overexpression of TIMP-1 in B16F10 cells reduces their metastatic ability in vivo by affecting tumor growth postextravasation.

Induction and expression of amphiregulin in human pancreatic cancer

Abstract: The epidermal growth factor receptor is activated by a family of polypeptides that includes the growth factor amphiregulin (AR). Using Northern blot analysis and the polymerase chain reaction, we now report that AR mRNA is expressed in human pancreatic cancer cell lines, and that this expression is enhanced in several of these cell lines by tetradecanoyl phorbol acetate and transforming growth factor α. AR was also expressed in normal and malignant pancreatic tissues. However, in the normal pancreas, AR immunostaining was most evident in the nuclei of ductal cells. In contrast, in many carcinomas, AR was also present in the cytoplasm of the ductal-like cancer cells. Cytoplasmic localization of AR was associated with a more advanced clinical stage. These findings suggest that AR may contribute to aberrant activation of the epidermal growth factor receptor in human pancreatic cancer, and may enhance disease progression.

Characterization of very acidic phagosomes in breast cancer cells and their association with invasion

Abstract: Human metastatic breast cancer cells in culture contain large acidic vesicles (diameter 5-10 microns) in which endocytosed extracellular matrix can be digested by activated lysosomal proteinases such as cathepsin D (P. Montcourrier et al. (1990). Cancer Res. 50, 6045-6054). We examined these large compartments by transmission electron microscopy, measured their pH by video-enhanced epifluorescence using FITC-dextran, and studied their functional significance. Their presence in metastatic MDA-MB231 cells was found to be correlated with an increased ability of cells to migrate through Matrigel and a high cathepsin D concentration. These cells were able to phagocytose 1.24 microns diameter latex beads and fluorescence Matrigel and incorporate this extracellular material into large acidic vesicles. This indicated that large acidic vesicles were associated with both phagocytosis and invasion, and are heterophagolysosomes rather than autophagosomes. Large acidic vesicles were actively acidified with a H(+)-ATPase vacuolar pump specifically inhibited by bafilomycin A1, and reached pH values (< 4), lower than the lysosomal value (pH approximately 5) in the same cells and in specialized phagocytotic cells such as macrophages. We conclude that the phagocytotic activity of breast cancer cells, associated with high cathepsin D expression, and high acidification potential, characterize cancer cells that have migrated through Matrigel.

Alterations in β-Catenin Phosphorylation and Plakoglobin Expression in Human Breast Cancer Cells

Abstract: Because the cell adhesion molecule epithelial cadherin (E-cadherin) is absent in many invasive carcinomas, we transfected the E-cadherin gene into E-cadherin-negative, invasive breast cancer cell lines BT549 and HS578t to investigate the role of E-cadherin in invasive behavior. Although the transfected E-cadherin could mediate calcium-dependent aggregation to E-cadherin-transfected L-cells, morphology and invasiveness of the breast cancer cells were not altered. We investigated the strength of the linkage of the transfected E-cadherin to the actin cytoskeleton by examining the Triton X-100 solubility of the transfected E-cadherin. In BT549 and HS578t cells, a large proportion of the transfected E-cadherin was Triton soluble, whereas in E-cadherin-positive MCF-7 cells, Triton-insoluble E-cadherin was apparent at cell-cell borders. Interaction of E-cadherin with the actin cytoskeleton is thought to be mediated by the E-cadherin-binding proteins alpha-catenin, beta-catenin, and plakoglobin. We found normal levels of alpha-catenin and beta-catenin in BT549 and HS578t cells; however, low levels of plakoglobin were expressed in these cells compared to those found in weakly invasive MCF-7 cells. Furthermore, levels of tyrosine phosphorylation of beta-catenin were elevated in E-cadherin-transfected BT549 and HS578t cells compared to MCF-7 cells. We conclude that other factors such as the expression and appropriate posttranslational modification of cadherin-associated proteins must be in place for E-cadherin to be fully functional, i.e., to alter invasiveness. During cancer progression, loss of E-cadherin expression itself or multiple other mechanisms that lead to loss of cell-cell adhesion (mutation, loss of catenin expression, alterations in phosphorylation) may contribute to a more metastatic phenotype.

The Urokinase Receptor Is Expressed in Invasive Breast Cancer but not in Normal Breast Tissue

Abstract: We have studied expression of the urokinase receptor (u-PAR) in paraffin-embedded breast tissues at various stages of malignant progression. Forty-nine of 59 invasive cancers studied showed varying degrees of reactivity with our polyclonal antibody. The staining pattern was variable from case to case, although strong surface staining of tumor-associated macrophages was evident in most of these sections. In several cases, blood vessels in selected tumor areas were stained, as confirmed by treatment of adjacent sections with an anti-factor VIII antibody. These could represent regions of recent angiogenesis. Staining of tumor cells was observed in 21 of 59 cases and was extensive in 5 cases but confined to a small percentage of cells in the remaining 16 samples. In contrast with the cancer sections, all normal breast tissue (12 cases) was negative, as well as all fibroadenomas (4 cases), papillomas (5 cases), and hyperplasia with atypia (2 cases) studied. Seven carcinomas in situ examined were also negative for u-PAR, with the exception of few macrophages in two cases, suggesting that u-PAR expression may be associated with invasive tumor. The presence of u-PAR in human breast cancer and its absence from nonmalignant breast tissue supports the idea that plasminogen activation plays an important role in the process of cancer invasion. Expression of u-PAR on macrophages, endothelial cells, and cancer cells suggests the existence of complex paracrine interactions between tumor cells and stroma.

The estrogen receptor from a tamoxifen stimulated MCF-7 tumor variant contains a point mutation in the ligand binding domain

Abstract: The nonsteroidal antiestrogen tamoxifen (TAM) is the most commonly used endocrine treatment for all stages of breast cancer in both pre- and postmenopausal women. However, the development of resistance to the drug is common, as most patients treated with TAM eventually experience a recurrence of tumor growth. One of the potential mechanisms of treatment failure is the acquisition by the tumor of the ability to respond to TAM as a stimulatory rather than inhibitory ligand. We (Gottardis and Jordan, Cancer Res 48: 5183-5187, 1988; Wolfet al., J Natl Cancer Inst 85: 806-812, 1993) and others (Osborneet al., Eur J Cancer Clin Oncol 23: 1189-1196, 1987; Osborneet al., J Natl Cancer Inst 83: 1477-1482, 1991) have extensively described the reproducible development of TAM stimulated growth in a laboratory model system using MCF-7 human breast cancer cells grown as solid tumors in athymic mice. In this paper we report on the isolation of an estrogen receptor (ER) from a TAM stimulated tumor (MCF-7/MT2) which contains a point mutation that causes a tyrosine for aspartate substitution at amino acid 351 in the ligand binding domain. The mutant appears to the major form of ER expressed by this tumor. We also report that only wild type ER was detected in three other TAM stimulated MCF-7 tumor variants, suggesting that multiple mechanisms are possible for the development of TAM stimulated growth. The implications of these findings are discussed.

Human prostate cancer cells: inhibition of proliferation by vitamin D analogs.

Abstract: 1,25-Dihydroxyvitamin D [1,25(OH)2D3, calcitriol] can inhibit the proliferation of some human prostate cancer cells but its clinical use is limited by hypercalcemia. We therefore explored the bioactivity of less calcemic vitamin D analogs. We studied the effects of calcitriol and 3 synthetic analogs at concentrations of 10(-6) to 10(-12) M on the in vitro proliferation of 3 human prostate carcinoma cell lines: DU 145, PC-3, and LNCaP. Calcitriol and analogs showed significant antiproliferative activity on PC-3 and LNCaP cells. DU 145 cells were inhibited by the analogs only. We conclude that vitamin D analogs warrant further investigation as therapeutic agents in prostate cancer.