Showing papers on "Cancer cell published in 1991"

Are cancer cells acidic

Abstract: For more than half a century it has been generally believed that cancer cells are more acidic than normal cells. This dogma arose from the studies of Warburg and co-workers (Warburg, 1930) who showed that tumour cells preferentially convert glucose and other substrates to lactic acid, even under aerobic conditions. Since lactic acid has a pK of 3.7 it seemed obvious that the intracellular fluid would become acidic. Studies on cultured tumour cells or spheroids often showed low intracellular pH (pHi) values, but this could have been due to their highly artificial situation. What would be the pHi of cells in a solid tumour in a patient? The development of pH electrodes small enough to be inserted into living tissues led to the apparent confirmation of the prevailing wisdom. Numerous studies (reviewed by WikeHooley et al., 1984 and Vaupel et al., 1989; see also Figure lb) showed significantly more acidic pH (pHpoT) in tumours than that in normal tissues. Microelectrodes that can be used on solid tumours in vivo are usually quite large in comparison to a tumour cell (see Wike-Hooley et al., 1984, Table II), and they mainly measure the pH of the extracellular fluid (pHe) rather than pHi (Vaupel et al., 1989). For most purposes, the parameter of interest is pH1, the pH of the water in the cancer cell itself, but it was generally expected (and then tacitly assumed) that pHi would also be acidic. This supposed cellular acidity in tumours became part of the mental wallpaper of oncologists and cancer researchers, even though there was, for many years, no practical way to measure pHi of intact human tumours. It had clinical consequences, too, since anticancer treatments were often designed to take advantage of a low pH, (for a review, see WikeHooley et al., 1984). It was argued, for instance, that anticancer drugs would be more effective if they contained ionising groups that would cause them to be trapped in acidic environments (Wike-Hooley et al., 1984), or that radioresistant hypoxic cells would have a particularly low pHi and might therefore be especially sensitive to treatments such as hyperthermia which are known to act preferentially on isolated cells in acidic media (Freeman et al., 1981). There were also numerous attempts to lower pHi still further by administration of glucose, and thereby enhance the action of various pH-sensitive therapies (Ross, 1961, reviewed by Wike-Hooley et al., 1984). In general, these ideas have had little clinical success, but they are still the subject of active research (see, for instance, Tannock & Rotin, 1989). Within the last 10 years a non-invasive intracellular pH meter the Nuclear Magnetic Resonance Spectrometer has become widely available; it can be used on living tumours in situ, both in experimental animals and in man. The results obtained with these instruments have been surprising. Instead of having the expected acidic pHi, the cells of intact tumours turned out to be neutral, or a little alkaline, both in experimental animals (Griffiths et al., 1981; Iles et al., 1982) and man (Griffiths et al., 1983). Indeed, several studies by 31P

Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin.

Abstract: Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in all types of cells, through several cycles and with minimal overall metabolic perturbations, has not yet been available. We describe a procedure for synchronizing normal as well as tumor cells reversibly in the G 1 phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This method of synchronization was successful with all cell lines tested, including normal and tumor cells of mouse, hamster, and human origins. For example, when MCF-7 human breast cancer cells were synchronized with Lovastatin and released by the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase), 3 phases of accelerated thymidine incorporation into DNA corresponding to 3 S phases of the cell cycle occurred during a 90-h period of cell replication. Thymidine incorporation was decreased to ≤4% during the initial lag of 18 h before the first S phase, and maximum incorporation was then achieved after only 6 h. The antibody Ki-67, which detects a nuclear antigen associated with proliferation, was present in cells arrested with Lovastatin. This fact, together with the lack of thymidine incorporation during the initial lag time, indicates that the cells were arrested in the G 1 and not in the G 0 phase of the cell cycle. Furthermore, in synchronized tumor-derived human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant synthesis of DNA and histone H4 RNA expression could be observed for 2 cycles. Minimal perturbations of general metabolic functions occurred since the rate of RNA, protein, and initial DNA synthesis were unaffected by Lovastatin, as evidenced by [ 3 H]uridine, [ 3 H]leucine, and initial [ 3 H]thymidine incorporation. Finally, while the Lovastatin-induced synchronization was overcome by mevalonic acid, addition of squalene or cholesterol-ethanol had no such effect. Thus, Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G 1 phase.

Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas.

Abstract: In this study in situ hybridization methods were used to examine biopsy samples from 13 adenocarcinomas of the colon for the presence of mRNA for the urokinase-type plasminogen activator (u-PA) and its specific cell-surface receptor (u-PAR). In all cases, u-PA mRNA was present in fibroblastlike cells in the stroma adjacent to the invasive tumor nodules. Urokinase-type plasminogen activator mRNA was not detected in the malignant cells. All specimens also contained u-PAR mRNA in cells located at the tumoral-stromal interface of invasive foci, but in contrast at least some of these cells were in all but one case identified as being of malignant origin. Stromal cells, probably tumor-infiltrating macrophages and neutrophils, also were positive in these areas. These results support the view that components of the plasminogen activation system may act to influence proteolytic events occurring at the interface between stroma and malignant cells in adenocarcinomas of the colon in humans.

Programmed cell death during regression of the MCF-7 human breast cancer following estrogen ablation.

Abstract: To study the mechanism of regression of human mammary cancer following estrogen ablation, estrogen-responsive MCF-7 human mammary adenocarcinoma cells were inoculated into ovariectomized female nude mice supplemented with exogenous 17 beta-estradiol (E2) via an E2 implant. Implants were then removed when MCF-7 tumors were 400 mm3 in size. Removal of the E2 implants resulted in a 50% tumor regression by 2 weeks following E2 ablation. Associated with this regression is a rapid (i.e., within 1 day following E2 ablation) enhanced expression of the transforming growth factor beta 1 and TRPM-2-genes, two genes the expression of which has been previously demonstrated to be enhanced in a variety of cell types induced to undergo programmed cell death (i.e., apoptosis). The enhanced expression of transforming growth factor beta 1 and TRPM-2 is not a nonspecific response since the expression of other genes, like c-fos, c-H-ras, and pS2, decrease following E2 ablation. Fragmentation of tumor DNA into nucleosomal oligomers and histological appearance of apoptotic bodies are characteristic early events that precede the dramatic reduction in tumor volume following E2 ablation. These results demonstrate that the regression of MCF-7 human mammary cancers in nude mice following estrogen ablation is due to a sequence of biochemical and morphological changes that result in both the cessation of cell proliferation and activation of programmed death or apoptosis of these MCF-7 cancer cells. Clarification of the biochemical pathway involved in the activation of this programmed cell death should identify new targets of therapy for even estrogen-independent human mammary cancer cells.

Tumor Cell Invasion Inhibited by TIMP-2

Abstract: The 72-kd type IV collagenase is a member of the collagenase enzyme family that has been closely linked with the invasive phenotype of cancer cells. Previous studies have shown that both normal cells and highly invasive tumor cells produce the 72-kd type IV procollagenase enzyme in a complexed form consisting of the proenzyme and a novel tissue inhibitor of metalloproteinases, TIMP-2. The balance between activated enzyme and available inhibitor is thought to be a critical determinant of the matrix proteolysis associated with a variety of pathologic processes, including tumor cell invasion. In the present study, we demonstrate that alteration of the metalloproteinase-metalloproteinase-inhibitor balance in favor of excess inhibitor blocks human fibrosarcoma HT-1080 tumor cell invasion of a reconstituted basement membrane. The HT-1080 cell line produces both the 72-kd and the 92-kd type IV collagenases. Alteration of the type IV collagenase-inhibitor balance was achieved by addition of free TIMP-2 or antibodies to 72-kd type IV collagenase. Native, purified TIMP-2 was inhibitory in the range of 1-25 micrograms/mL. Addition of specific antiserum against the 72-kd type IV collagenase, which did not cross-react with the 92-kd type IV collagenase, inhibited HT-1080 cell invasion to the same extent. These results suggest that metalloproteinases, in particular the 72-kd type IV collagenase, are critical for tumor cell invasion of the reconstituted basement membrane. Our findings demonstrate that addition of the endogenous inhibitor TIMP-2 is able to block invasion. Thus, we recommend initiation of in vivo studies of the therapeutic potential of TIMP-2 to block tumor cell invasion and intravasation into the circulation.

Relationship of the Expression of the Multidrug Resistance Gene Product (P-Glycoprotein) in Human Colon Carcinoma to Local Tumor Aggressiveness and Lymph Node Metastasis

Abstract: P-glycoprotein mediates classic multidrug resistance by functioning as an efflux pump that excretes lipophilic chemotherapeutic drugs from cancer cells. We now report an association of P-glycoprotein in colon carcinomas with another tumor property, i.e. , enhancement of local tumor aggressiveness. P-glycoprotein was detected with monoclonal antibody immunohistochemistry in 65 of 95 primary colon adenocarcinomas, which were stage B1 or greater. In all but 1 of the 95 cases, solitary invading carcinoma cells were present at the leading edge of the tumor. This subpopulation of invasive carcinoma cells expressed P-glycoprotein (P-Gp+) in 47 of the 95 surgically resected colon specimens. Cases were grouped on the basis of the presence (Group 1, 47 cases) or absence (Group 2, 48 cases) of P-Gp+ invasive carcinoma cells. There was a significantly greater incidence of vessel invasion ( P 0.1). Our findings indicate that P-Gp+ invasive colon cancer cells may have an increased potential for dissemination, suggesting that P-glycoprotein may influence cell behavior.

Growth suppression of human breast cancer cells by the introduction of a wild-type p53 gene.

Abstract: Mutations in the p53 gene are associated with a wide variety of human tumors, including those of the breast. To assess functionally the role of the p53 gene in the development of human breast cancer, we introduced either wild-type or mutant p53 cDNA into three human breast cancer cell lines by DNA transfection. The cell lines MDA-MB 468 and T47 D contain only single mutated copies of the p53 gene, whereas the status of p53 in the breast cancer cell line MCF 7 remains equivocal. Following transfection, MCF 7 cells continued to grow unaffected both in vitro and in vivo in the presence of high levels of expression of the exogenous wild-type p53 gene. In contrast, however, the continued expression of an exogenous wild-type p53 gene was incompatible with cellular growth in both the MDA-MB 468 and T47 D cell lines. Elevated levels of expression of the exogenous mutant p53 gene did not alter the growth of the cell lines in vitro. These data strongly suggest that the wild-type p53 gene can function as a suppressor of cellular growth in breast cancer cells. That the wild-type p53 gene does not suppress the growth of MCF 7 cells indicates that at least some human breast tumors can arise without functional inactivation of the p53 gene by mutation. These tumors may represent a separate prognostic group.

Cell-cycle-related staining patterns of anti-proliferating cell nuclear antigen monoclonal antibodies. Comparison with BrdUrd labeling and Ki-67 staining.

Abstract: Monoclonal antibodies (MAbs) to nuclear antigens are increasingly used as tools to obtain valuable information concerning the proliferative characteristics of various types of cancer. Prerequisite for the application of these MAbs in surgical pathology is establishment of the level of expression and/or cellular distribution of the antigens in relation to distinct cell-cycle compartments. In this study the topologic distribution of proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase delta, as recognized by human autoantiserum (AK) and two recently developed MAbs (19A2 and 19F4), was evaluated. Using cultured human cancer cells as a model system, and providing optimal fixation/permeation procedures are applied, these antibodies display a high affinity for PCNA bound to nuclear replicon clusters, resulting in distinct granular staining patterns. A more diffuse nucleoplasmic PCNA staining was mainly restricted to non-S-phase cells; in methanol-fixed cells, staining intensity of this form relative to the replicon-bound form appeared higher after staining with 19A2 than with 19F4 or AK. Comparing PCNA expression (detected with 19A2) with the expression of the Ki-67 antigen, PCNA-negative cells are also Ki-67 negative. In MCF-7 human breast cancer cells treated with 10(-6) mol/l (molar) tamoxifen, the fraction of nuclei showing replication patterns decreased from 42% to 8% within 8 days, but PCNA and Ki-67 antigens remained detectable in most cells during this interval, indicating a relatively slow decrease of antigen expression in cells that have entered a quiescent state. Treatment of MCF-7 cells with 10(-6) mol/l methotrexate resulted in a rapid accumulation of cells with an early S-phase DNA content; PCNA replication patterns showing a frequency distribution reflecting this DNA content were observed up to 48 hours after treatment. This indicates that the presence of replication patterns as visualized with anti-PCNAs is not a measure of replicative activity per se. It is concluded that, providing nuclear non-S-phase PCNA staining is faint relative to staining of replicon clusters, anti-PCNA antibodies may be excellent markers to detect in situ cells with S-phase DNA contents.

Effects of fatty acids and eicosanoid synthesis inhibitors on the growth of two human prostate cancer cell lines.

Abstract: Dietary fatty acids (FAs) may be involved in the carcinogenic process within the prostate gland and progression to clinically manifest disease. We have shown that growth of the androgen-unresponsive PC-3 human prostate cancer cell line is stimulated in vitro by the presence of linoleic acid (LA), an omega-6 polyunsaturated FA. The response was positively related to the FA concentration over the entire range examined (5-750 ng/ml). Conversely, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), two omega-3 FAs present in fish oils, inhibited PC-3 cell growth in a dose-dependent manner; both were equally effective, with an approximately 65% reduction in growth occurring at a concentration of 2.0 micrograms/ml (P less than 0.001). The DU 145 human prostate cancer cell line, which is also androgen-unresponsive, showed no growth response to LA and was less susceptible to growth inhibition when cultured in the presence of omega-3 FAs. Growth experiments with indomethacin, esculetin, and piroxicam, pharmacological inhibitors of eicosanoid biosynthesis with differing sites of action, indicated that human prostate cancer cell growth requires intact metabolic pathways for both leukotriene and prostaglandin production.

An additional homolog of the fission yeast cdc25+ gene occurs in humans and is highly expressed in some cancer cells.

Abstract: The gene cdc25+ is a mitotic inducer controlling transition from the G2 to the M phase of the cell cycle in the fission yeast, Schizosaccharomyces pombe. Using phenotypic complementation of a mutant of S. pombe, we have cloned a human homolog (CDC25Hu2) of the cdc25+ gene that differs markedly in structure from CDC25 (referred to here as CDC25Hu1), the first such homolog to be isolated. The carboxyl-terminal region of p63CDC25Hu2 shares significant sequence similarity with cdc25 protein homologs from other eukaryotes and possesses full complementation activity. CDC25Hu2 is expressed in human cell lines 10 to 100 times more than CDC25Hu1, and its expression is particularly high in some cancers, including SV40-transformed fibroblasts. Whereas CDC25Hu1 is predominantly expressed in G2, CDC25Hu2 is expressed throughout the cell cycle with a moderate increase in G2. Thus, at least two homologs of the cdc25 gene exist and are both expressed in human cells. The implications of CDC25Hu2 overexpression in some cancer cells are discussed.

The antiproliferative effects of tyrosine kinase inhibitors tyrphostins on a human squamous cell carcinoma in vitro and in nude mice

Abstract: Many human tumors of epithelial origin contain cells overexpressing the epidermal growth factor (EGF) receptor, and there is convincing evidence that cancer cell growth is correlated with the loss of the normal regulation of the EGF receptor signal transduction pathway. Some cancers are clearly dependent on activation of the EGF receptor for their proliferation. Recently, a class of compounds, tyrphostins, which inhibit the protein tyrosine kinase activity of the growth factor receptor, have been described. In this report, we have examined the antiproliferative effects of potent new tyrphostins on a well-characterized human squamous cell carcinoma in vitro and in vivo. We found that two of these compounds (RG-13022 and RG-14620) suppressed not only EGF-stimulated cancer cell proliferation in vitro but also tumor growth in nude mice. RG-13022 also increased the life span of these tumor-bearing nude mice. When administered to tumor-bearing nude mice together with monoclonal antibodies to the EGF receptor at a suboptimal dose which had no effect alone, inhibition of tumor growth was markedly enhanced. These data suggest that tyrphostins have potential as anticancer agents.

Programmed death of nonproliferating androgen-independent prostatic cancer cells.

Abstract: Androgen ablation induces an energy-dependent process of programmed death in nonproliferating androgen-dependent prostatic cancer cells which involves fragmentation of genomic DNA into nucleosomal oligomers catalyzed by nuclear Ca2+, Mg(2+)-dependent endonuclease enzymes activated following a sustained elevation in intracellular free Ca2+ (Cai). In contrast, androgen-independent prostatic cancer cells are not induced to undergo such programmed cell death by androgen ablation. One explanation for the inability of androgen ablation to induce programmed death of androgen-independent prostatic cancer cells is that such ablation does not result in a sustained elevation in Cai in these cells. This raises the issue of whether androgen-independent prostatic cancer cells can be induced to undergo programmed death if an elevation in the Cai is sufficiently sustained by nonhormonal means. To test this possibility, androgen-independent, highly metastatic Dunning R-3327 AT-3 rat prostatic cancer cells were chronically exposed in vitro to the calcium ionophore ionomycin to sustain an elevation in their Cai. These studies demonstrated that an elevation of Cai as small as only 3-6-fold above baseline can induce the death of these cells if sustained for greater than 12 h. Temporal analysis demonstrated that the death of these cells does not require cell proliferation and involves Ca(2+)-induced fragmentation of genomic DNA into nucleosome-sized pieces as the commitment step in this process. These results demonstrate that even nonproliferating androgen-independent prostatic cancer cells can be induced to undergo programmed cell death if a modest elevation in the Cai is sustained for a sufficient time. These observations identify Cai as a potential target for therapy for androgen-independent prostatic cancer cells.

Effects of melatonin on the cell cycle kinetics and “estrogen-rescue” of MCF-7 human breast cancer cells in culture

Abstract: Melatonin has been shown to have a direct inhibitory action on the proliferation of estrogen-responsive MCF-7 human breast cancer cells in culture. In the present study, we examined by flow cytometry whether this inhibitory effect might be exerted on the G1 phase of the cell cycle, thus causing a transition delay into the S phase. In order to further verify this hypothesis we tested the ability of estradiol to "rescue" MCF-7 cells from melatonin inhibition, and the potential of this indoleamine to block the ability of estradiol to rescue the cells from tamoxifen inhibition. Following five days of incubation, melatonin (10(-9)M) increased the fraction of cells in G1 of the cell cycle while simultaneously causing a 50% reduction in the proportion of cells in S phase. The antiproliferative effect of melatonin (10(-5)M) was prevented by the simultaneous treatment of the cells with estradiol (10(-8)M) in clonogenic soft agar culture, or reversed by the addition of estradiol to cells previously incubated with and inhibited by melatonin (10(-9)M) in monolayer culture. Additionally, melatonin blocked the estrogen-rescue of tamoxifen-inhibited cells in both types of culture systems. These results support the hypothesis that the antiproliferative effect of melatonin, like tamoxifen, is cell cycle specific by causing a G1-S transition delay. These results also indicate an important interaction of melatonin with estrogen-mediated mechanisms of MCF-7 cell proliferation.

Changes in expressions of proteasome and ubiquitin genes in human renal cancer cells.

Abstract: Proteasomes and ubiquitin (Ub) are essential components of the energy-dependent, nonlysosomal proteolytic pathway. To clarify the physiological role of this proteasome/Ub-dependent pathway, we meaured the levels of expressions of proteasomes and Ub in human renal cancers by Northern blot and immunochemical analyses. The mRNAs for two of the multiple subunits of proteasomes, C2 and C9, were expressed at abnormally high levels in most neoplastic lesions of patients with various primary renal cell carcinomas and in all renal cancer cell lines examined. However, no significant difference was found by enzyme immunoassay in the proteasomal contents of cancerous and normal parts of the kidney. The levels of mRNAs for the subunits of proteasomes were high in rapidly proliferating renal cells and appeared to be correlated with the activities of these cells for proteasome synthesis, but the cellular contents of proteasomes in these cells were normal, suggesting rapid turnover of proteasomes in rapidly proliferating cancer cells. Consistent with the increased expressions of proteasomal mRNAs, the expressions of three Ub genes, mono-UbA80, mono-UbA52, and poly-UbC, were found to be greatly increased in these renal cancer cells. Immunohistochemical staining of normal kidney showed that the levels of both proteasomes and Ub were high in cells of renal tubules and collecting ducts, but low in the glomerulus. The levels of both proteins appeared to be considerably increased in the nuclei of granular and clear carcinoma cells of the kidney. Moreover, the profiles of cellular proteins conjugated with Ub in normal kidney tissues were different from those in cancerous parts of the kidney and in established renal cancer cells. These results suggest that the proteasome- and ubiquitin-mediated system is functionally involved in the cancerous state in human kidney.

The Plasminogen Activation System in Human Colon Cancer: Messenger RNA for the Inhibitor PAI-1 Is Located in Endothelial Cells in the Tumor Stroma

Abstract: Fourteen human colon adenocarcinomas were examined by in situ hybridization for the presence of mRNA for plasminogen activator inhibitor type 1 (PAI-1). All specimens contained PAI-1 mRNA in endothelial cells of some vessels in the stroma immediately surrounding the invasive tumor glands, in granulation tissue, and in some capillaries located under the free luminal surface of carcinomatous epithelium. In addition, a limited number of stromal cells in the cancerous areas located at the periphery of newly formed capillary networks, and presumably representing sprouting endothelial cells, contained PAI-1 mRNA. Cancer cells were devoid of detectable PAI-1 mRNA in all cases. PAI-1 was not seen in three biopsies of normal colon. Together with previous findings of urokinase-type plasminogen activator and its mRNA being located in fibroblast-like cells in the tumor stroma and mRNA for the urokinase receptor in the cancer cells at invasive foci, these results indicate a complex cooperativity among several cell types in regulation of plasminogen activation in colon cancer. A possible role of PAI-1 in protecting the extracellular matrix in the tumor tissue against degradation and a role in tumor-induced angiogenesis are discussed.

Mediation of lung metastasis of murine melanomas by a lung-specific endothelial cell adhesion molecule.

Abstract: Organ-specific adhesion molecules expressed by vascular endothelial cells have been implicated in the arrest of blood-borne cancer cells in selective, secondary sites. A lung-specific endothelial cell adhesion molecule (Lu-ECAM-1) localized on endothelia of distinct branches of lung blood vessels has been purified by immunoaffinity chromatography from detergent extracts of lung matrix-modulated endothelial cells using monoclonal antibody (mAb) 6D3. It has a molecular mass of 90 kDa and promotes the selective attachment of lung-metastatic B16 melanoma cells. Corresponding with their metastatic performance, B16-F10 tumor cells selected for higher lung colonization bind to Lu-ECAM-1 in significantly higher numbers than their low lung metastatic counterpart B16-F0. Binding of B16-F0 and B16-F10 is reduced with mAb 6D3 to slightly lower levels than B16-F0 bound to Lu-ECAM-1. mAb 6D3 injected into C57BL/6 mice 1 hr prior to an i.v. challenge with B16-F10 causes a 90% reduction in the number of lung colonies compared with animals injected with control mAb (6D8 or 3C6). Lu-ECAM-1 neither binds nor effects metastasis of other lung-colonizing tumor cells (e.g., KLN205). Thus, site-specific metastasis of tumor cells is regulated by similar mechanisms as the homing of lymphocytes--namely, by the ability of blood-borne cancer cells to recognize and adhere to distinct endothelial cell adhesion molecules.

Selective analysis of antitumor drug interaction with living cancer cells as probed by surface-enhanced Raman spectroscopy.

Abstract: A new technique for the selective measurement of small amounts of antitumor drugs in the nucleus and cytoplasm of a living cancer cell, based on surface-enhanced Raman spectroscopy (SERS), is proposed. The ability to detect SERS signals from very dilute (up to 10−10 M) solutions of doxorubicin or adriamycin (DOX), and 4′O-tetrahydropyranyl-adriamycin (THP-ADM), as well as from their complexes with targets in vitro and in vivo, has been demonstrated. SERS spectra were obtained from a population as well as from single living erythroleukaemic K562 cells treated with DOX. The results of the measurements on the population of cells containing DOX in nuclei or in the cytoplasm are well correlated with the microscopic SERS measurements on the single cells treated with DOX, obtained by selectively recording signals from the living cell nucleus or from the cytoplasm. Possibilities for the application of this new technique in different aspects of cancer research are discussed.

Cell membrane signaling as target in cancer therapy: inhibitory effect of N,N-dimethyl and N,N,N-trimethyl sphingosine derivatives on in vitro and in vivo growth of human tumor cells in nude mice.

Abstract: Sphingosine (SPN) has been claimed to be a negative modulator of transmembrane signaling through protein kinase C (PK-C) or some yet unidentified mechanism [for review see Y. A. Hannun and R. M. Bell, Science (Washington DC), 243: 500–507, 1989]. N,N -Dimethylsphingosine (DMS) was recently found to be a physiological cellular component and, in comparison to SPN, to show a stronger and stereospecific inhibitory effect on PK-C activity of A431 cells (for review see Y. Igarashi, Trends Glycosci. Glycotechnol., 2: 319–332, 1990; and S. Hakomori, J. Biol. Chem., 265: 18713–18716, 1990). (4 E )- N,N,N -Trimethyl-d- erythro -sphingenine (TMS) is not detectable as a normal cellular component; however, it is expected to exhibit potent activity because of its quaternary ammonium ion structure, and in fact it showed much stronger inhibitory effect than DMS or SPN on PK-C activity (which plays an important role in cell growth regulation) in vitro . In view of these findings, we investigated the effects of SPN, DMS, and TMS on in vitro growth of various human carcinoma cell lines and on in vivo tumor growth in athymic nu/nu mice. Both DMS and TMS showed similar in vitro and in vivo growth inhibitory effects on tumor cells, despite the fact that TMS showed a much stronger inhibitory effect than DMS on PK-C activity of A431 cells. In contrast, SPN showed only a weak effect on in vitro cell growth and no effect on in vivo tumor growth. Tumor growth following s.c. inoculation of mice with human gastric carcinoma cell line MKN74 was inhibited in a dose-dependent manner by DMS, and tumor size was decreased after three or four consecutive daily injections of 0.5-mg doses of DMS or TMS. Increased tumor growth occurred after administration of these compounds was stopped; however, size of tumor remained significantly smaller than in groups treated with SPN or control saline. The effect of DMS or TMS on in vitro or in vivo MKN74 cell growth was stronger than that of 8-chloro-adenosine-cyclic 3′:5′-monophosphate dihydrate, the most promising agent currently being used in clinical trials for inhibition of tumor growth by induction of differentiation. These results suggest that DMS or TMS could be useful anticancer agents through modification of transmembrane signaling related to cancer cell growth.

Haemodynamic and transport barriers to the treatment of solid tumours.

Abstract: The efficacy in cancer treatment of novel therapeutic agents such as monoclonal antibodies, cytokines and effector cells has been limited by their inability to reach their target in vivo in adequate quantities. Molecular and cellular biology of neoplastic cells alone has failed to explain the nonuniform uptake of these agents. This is not surprising since a solid tumour in vivo is not just a collection of cancer cells. In fact, it consists of two extracellular compartments: vascular and interstitial. Since no blood-borne molecule or cell can reach cancer cells without passing through these compartments, the vascular and interstitial physiology of tumours has received considerable attention in recent years. Three physiological factors responsible for the poor localization of macromolecules in tumours have been identified: (i) heterogeneous blood supply, (ii) elevated interstitial pressure, and (iii) large transport distances in the interstitium. The first factor limits the delivery of blood-borne agents to well-perfused regions of a tumour; the second factor reduces extravasation of fluid and macromolecules in the high interstitial pressure regions and also leads to an experimentally verifiable, radially outward convection in the tumour periphery which opposes the inward diffusion; and the third factor increases the time required for slowly moving macromolecules to reach distal regions of a tumour. Binding of the molecule to an antigen further lowers the effective diffusion rate by reducing the amount of mobile molecule. Although the effector cells are capable of active migration, peculiarities of the tumour vasculature and interstitium may also be responsible for poor delivery of lymphokine activated killer cells and tumour infiltrating lymphocytes in solid tumours. Due to micro- and macroscopic heterogeneities in tumours, the relative magnitude of each of these physiological barriers would vary from one location to another and from one day to the next in the same tumour, and from one tumour to another. If the genetically engineered macromolecules and effector cells, as well as low molecular weight cytotoxic agents, are to fulfill their clinical promise, strategies must be developed to overcome or exploit these barriers. Some of these strategies are discussed, and situations wherein these barriers may not be a problem are outlined. Finally, some therapies where the tumour vasculature of the interstitium may be a target are pointed out.

The growth inhibition of human breast cancer cells by a novel synthetic progestin involves the induction of transforming growth factor beta.

Abstract: Recent experimental work has identified a novel intracellular binding site for the synthetic progestin, Gestodene, that appears to be uniquely expressed in human breast cancer cells. Gestodene is shown here to inhibit the growth of human breast cancer cells in a dose-dependent fashion, but has no effect on endocrine-responsive human endometrial cancer cells. Gestodene induced a 90-fold increase in the secretion of transforming growth factor-beta (TGF-beta) by T47D human breast cancer cells. Other synthetic progestins had no effect, indicating that this induction is mediated by the novel Gestodene binding site and not by the conventional progesterone receptor. Furthermore, in four breast cancer cell lines, the extent of induction of TGF-beta correlated with intracellular levels of Gestodene binding site. No induction of TGF-beta was observed with the endometrial cancer line, HECl-B, which lacks the Gestodene binding site, but which expresses high levels of progesterone receptor. The inhibition of growth of T47D cells by Gestodene is partly reversible by a polyclonal antiserum to TGF-beta. These data indicate that the growth-inhibitory action of Gestodene may be mediated in part by an autocrine induction of TGF-beta.

Identification of High Mobility Group Protein I(Y) as Potential Progression Marker for Prostate Cancer by Differential Hybridization Analysis

Abstract: One of the major problems in the diagnosis of localized prostatic tumors is to predict the aggressiveness of an individual tumor, which is presumably associated with chance to progression. In an attempt to find molecular markers that are specific for aggressive prostatic cancer cells, we compared steady-state mRNA levels of progressionally related prostatic tumors. The Dunning R-3327-H subline, a relatively benign rat prostatic tumor, was compared to the therefrom derived highly aggressive MatLyLu tumor by differential hybridization analysis. The differential screening revealed 26 complementary DNA clones that detected transcripts overexpressed in MatLyLu. Upon further screening on the entire panel of Dunning R-3327 sublines, it appeared that three clones (pBUS1, pBUS19, and pBUS30), detected transcripts specifically expressed in metastatic rat prostatic tumors. The expression pattern of pBUS19 and pBUS30 suggested a relation between these complementary DNAs. Nucleotide sequence analysis, however, could not yet substantiate this. Computer-assisted comparison of the DNA sequences revealed the presence of rat long terminal repeat-like repetitive elements in pBUS19. The differential expression of repetitive elements in progressionally related tumors is interesting, yet similar findings have not been reported in human malignancies. Nucleotide sequence analysis of pBUS1 indicated that this clone is identical or related to high mobility group protein I(Y), a non-histone nuclear protein. From recent studies it appeared that this protein might be implicated in replication and/or transcription processes and is induced in fast proliferating/undifferentiated cells. The overexpression of high mobility group protein I(Y) correlates rather with metastatic ability than with growth rate; hence it may serve as a valuable marker to identify progressionally advanced prostate cancer cells.

Characteristics of the Biphasic Action of Androgens and of the Potent Antiproliferative Effects of the New Pure Antiestrogen EM-139 on Cell Cycle Kinetic Parameters in LNCaP Human Prostatic Cancer Cells

Abstract: The most potent steroid in human prostatic carcinoma LNCaP cells, i.e., dihydrotestosterone (DHT), has a biphasic stimulatory effect on cell proliferation. At the maximal stimulatory concentration of 0.1 nM DHT, analysis of cell kinetic parameters shows a decrease of the G0-G1 fraction with a corresponding increase of the S and G2 + M fractions. In contrast, concentrations of 1 nM DHT or higher induce a return of cell proliferation to control levels, reflected by an increase in the G0-G1 fraction at the expense of the S and especially the G2 + M fractions. Continuous labeling for 144 h with the nucleotide analogue 5'-bromodeoxyuridine shows that the percentage of cycling LNCaP cells rises more than 90% after treatment with stimulatory concentrations of DHT, whereas in control cells as well as in cells treated with high concentrations of the androgen, this value remains below 50%. Although LNCaP cells do not contain detectable estrogen receptors, the new pure steroidal antiestrogen EM-139 not only reversed the stimulation of cell proliferation and cell kinetics induced by stimulatory doses of DHT but also inhibited basal cell proliferation.

Sensitivity of Immunocytochemical Detection of Breast Cancer Cells in Human Bone Marrow

Abstract: We have previously shown that occult micrometastases can be detected in the bone marrow of breast cancer patients, at the time of initial treatment, using a panel of epithelial specific monoclonal antibodies indirectly labeled with fluorescein. These monoclonal antibodies permit us to detect cancer cells at a concentration of two/million normal bone marrow cells. Immunofluorescence carries the disadvantage that detailed morphological examination of detected cells cannot be accomplished. A modification of the alkaline phosphatase anti-alkaline phosphatase method has been used to detect cancer cells and to observe their morphology in human bone marrow. The sensitivity of this method has been examined using an established human metastatic breast cancer cell line (MCF-7) mixed with normal bone marrow cells at various dilutions from 400 cancer cells/10 6 marrow cells to 10 cancer cells/10 6 marrow cells. The number of immunocytochemically stained MCF-7 cells counted at each concentration was related to the concentration by a simple nonlinear statistical model. At a concentration of 10 cancer cells/10 6 bone marrow cells, the model shows that this method has the sensitivity to detect between four and six MCF-7 cells 95% of the time. Extrapolation, using this model, predicts that at the very low concentration of one cancer cell/10 6 marrow cells, there is a 95% chance of detecting the cancer cell. This assay may be a very sensitive method for detecting cancer cells in the bone marrow of breast cancer patients.

Detection of mRNAs encoding collagenase I and stromelysin 2 in carcinomas of the head and neck by in situ hybridization.

Abstract: The mRNAs encoding 2 metalloproteinases, stromelysin 2 and collagenase I, have been detected by in situ hybridization in 26 carcinomas of the head and neck. 23 tumors of 26 expressed these mRNAs. Collagenase mRNAs were present in individual invasive cancer cells and in tumor cells at the periphery of poorly differentiated clusters (4 cases). Numerous stromal cells, principally fibroblasts were labeled (18 cases). Stromelysin mRNAs have been localized in tumor cells frequently arranged along disrupted basement membranes (8 cases). Many stromal cells in close contact to cancer cells also expressed the stromelysin mRNAs (17 cases). Normal residual cells were never labeled. These observations plead for the role of stromelysin produced by both stromal and tumor cells in the breakdown of basement membranes and the involvement of both collagenase and stromelysin in stromal invasion in carcinomas of the head and neck in vivo.

Inhibition of human breast cancer cell (MCF-7) growth in vitro by the somatostatin analog SMS 201-995: effects on cell cycle parameters and apoptotic cell death.

Abstract: Somatostatin (SS) and SS analogs have been shown to exert an antiproliferative effect on several transplantable tumors in animals and to reduce the growth of pancreatic, pituitary, and mammary tumor cells in vitro. We evaluated the effects that the SS analog SMS 201-995 exerts on growth, cell-cycle parameters, and suicidal cell death (apoptosis) of human breast cancer cells (MCF-7) in vitro. SMS 201-995 significantly reduced the MCF-7 cell growth induced by serum, estradiol, insulin, and insulin-like growth Factor-I in both short term and long term experiments. The effect was maximal when 10 nM estradiol was used as mitogen in long term cultures. SMS 201-995 treatment produced a slight but transient accumulation of cells in the G2/M phase but did not cause any noteworthy reduction in the percentage of proliferating cells. There was, instead, a time-related increase in the number of cells with the flow-cytometric characteristics of apoptosis in the cultures treated with the SS analog, which correlated well with its growth-inhibiting activity. It would, therefore, seem that SMS 201-995 exerts its inhibitory effect on MCF-7 cell growth in vitro mainly by enhancing the rate of programmed (or suicidal) cell death in the culture.