[Bcl-2 family proteins and mechanisms of the acceleration and inhibition of tumor progression in vivo].

Earlier the authors demonstrated that the process of tumor progression in vivo may be inhibited or accelerated depending on the conditions of tumor growth (accelerated by tumor cell dissemination or delayed in locally growing tumors). It was also shown that tumor progression is inhibited in case of bcl-2 gene transduction in tumor cells. In this study, the research into mechanisms of the acceleration or inhibition of tumor progression and the role that Bcl-2 family proteins may play in these phenomena was continued. The results of the study demonstrated the following 1) immediate in vivo activation of endogenous proapoptotic Bax protein in disseminated tumor cells, not protected by Bcl-2 against apoptosis, and its correlation with accelerated tumor progression; 2) complete suppression of in vivo Bax activation in tumor cells protected by Bcl-expression, and inhibited tumor progression; 3) alternative character of Bcl-2 and Bax expression under the conditions of accelerated and inhibited tumor progression. Thus, the data presented support the hypothesis that the rates of tumor progression in vivo are regulated depending on the initial anti- and proapoptotic programs of tumor cells.

Changes in the balance between membrane-bound and soluble forms of CD95 (Fas) during selection of tumor cells in vivo.

Studies concerning the expression of the receptor CD95 (Fas) by tumor cells and the role of this protein in apoptosis induced by the effector host cells that bear Fas-ligand are mainly focused on the membrane-bound form of Fas. There are only a few data about the production of the soluble form of Fas by the tumor cells, its role in the interaction with the effector host cells, and the possible changes in the synthesis of this protein during tumor progression. In the present work, three in vitro transformed parental cell lines of different origin and 24 of their variants isolated after a short cycle of natural selection in vivo were studied. It was demonstrated for the first time that: 1) production of the soluble Fas by all selected in vivo variant tumor cell lines increased significantly (2-10-fold) in comparison to the initial (parental) cell lines and did not depend on the origin of the parental lines. At the same time, the expression of the membrane-bound form of Fas decreased considerably; 2) variations of the balance between membrane-bound and soluble forms of Fas in selected in vivo variant cells and the expression of the [H(2)O(2)(CA) + PGE(S)]-phenotype by these cells (this phenotype determines one of the essential mechanisms of the protection of a tumor cell in vivo) possibly represent independent secondary changes acquired during tumor progression in vivo.

Natural selection and early changes of phenotype of tumor cells in vivo: acquisition of new defense mechanisms.

This review summarizes results obtained in the author's and collaborating laboratories within the last decade and is designed to attract the attention of researchers to discrete biochemical mechanisms of protection acquired in vivo by cells of malignant tumors against effectors of innate antitumor immunity. Tumor progression in vivo is associated with the appearance and selection of tumor cells with new specific characteristics: a high level of H(2)O(2)-catabolizing (antioxidant) activity (H(2)O(2)CA) and the ability for immediate release of E2-type prostaglandin (PGES) on contact with natural killers, macrophages, and neutrophils; the expression of the [H(2)O(2)CA + PGES] phenotype provides the tumor cells with two mechanisms of local protection against effectors of innate and acquired antitumor immunity. This results in a 10-100-fold less effective rejection of tumor cells in immune and normal animals and corresponding increase of tumorigenicity. The in vitro transformation of normal fibroblasts, spontaneous or induced by oncogenes LTSV40, E1a,b, Ha-ras, myc, and also by p53(175) and bcl-2 does not result in the [H(2)O(2)CA + PGES] phenotype expression, but during subsequent in vivo growth of the above-mentioned transformants the selection of tumor cells of the [H(2)O(2)CA + PGES] phenotype is correlated with a 30-200-fold increase in their tumorigenicity (accompanied or not accompanied by spontaneous metastatic activity). Unlike the transformation induced by the above-mentioned oncogenes, the transformation of normal cells by the v-src gene results in the [H(2)O(2)CA + PGES] phenotype expression. The data presented confirm the determining role of the v-src gene in the expression of the [H(2)O(2)CA + PGES] phenotype. In various primary viral carcinogenesis (SV40, SA7(C8)) the natural selection of cells expressing the [H(2)O(2)CA + PGES] phenotype begins even within the latent period and can be completed by the appearance of primary tumors.

Nonactivated macrophage-induced nitric oxide production by tumor cells differing in tumorigenic and spontaneous metastatic activities.

The ability of nonactivated peritoneal macrophages to induce nitric oxide (NO) secretion in transformed and tumor cells of the same origin differing in tumorigenic (TGA) and spontaneous metastatic activities (SMA) was examined. Low tumorigenic and nonmetastatic spontaneously transformed in vitro hamster embryo cells of the STHE strain in contact with macrophages, or their non-purified soluble product were the highest producers of NO. In vivo selected STHE cell variants characterized by middle or high TGA demonstrated low, or no NO production, respectively (independently of SMA- or SMA+). Two highly tumorigenic RSV-SR (v-src) transformed cell strains (SMA- and SMA+) were both negative in NO production. Thus, NO production by tumor cells appeared to be inversely correlated with TGA level and less dependent on SMA.

Suppression of apoptosis by bcl-2 does not prevent p53-mediated control of experimental metastasis and anchorage dependence.

Mutations in the p53 tumor suppressor gene are frequently associated with the metastatic stage of tumor progression. Inactivation of p53 was shown to promote metastasis under experimental conditions. To determine the p53 functions that are involved in the control of tumor metastasis, we compared properties of three types of transformed mouse fibroblasts: with intact p53, with p53-mediated apoptosis suppressed by bcl-2 and with p53 inactivated by dominant negative mutants. Although expression of bcl-2 blocked apoptosis in detached cells and increased tumor cell survival in the blood circulation, it was insufficient to affect the ability of p53 to cause cell cycle arrest in detached cells and suppress experimental metastasis. For the suppression of metastasis complete inactivation of p53 was required. We conclude that the apoptotic function of p53 is dispensable for the p53-dependent suppression of experimental metastasis that is presumably achieved by controlling anchorage dependence. These data provide a possible explanation to dramatic differences in values of bcl-2 and mutant p53 as prognostic markers in human cancer.

p53 modulation of anchorage independent growth and experimental metastasis.

Death in circulation is one of the natural barriers preventing dissemination of tumor cells and formation of metastases. One of the negative factors acting in circulation is the loss of cell contact with natural substrate which can be imitated in vitro by the incubation of cells in suspension or in semi-solid media. Normal mouse fibroblasts (MEFs) stay viable in suspension and undergo p53-independent G1 growth arrest. Transformation with Ela and ras oncogenes leads to the abrogation of this arrest and to the p53-dependent apoptosis occurring in G1 phase of the cell cycle. Suppression of apoptosis by p53 gene knock-out, transduction of dominant negative p53 mutant or bcl-2 prevents death in suspension and greatly induces frequency of colony formation in semi-solid media. The ability of cells to undergo apoptosis does not correlate with their tumorigenicity in nude mice but does correlate with their ability to survive in lungs of intravenously injected mice and to form experimental metastases. We suggest that abrogation of a p53-mediated apoptosis facilitates experimental metastasis by promoting survival of tumor cells in circulation.

Mechanisms of unusually high antioxidant activity of RSV-SR-transformed cells and of its suppression by activated p21ras.

We have previously demonstrated that hamster embryo fibroblasts (HEFs) transformed by Rous Sarcoma virus, Schmidt-Ruppin strain (RSV-SR) are highly resistant to damage by H202 (H2O2R), (in contrast to HEFs transformed spontaneously, or by bovine adenovirus and SV40), while N-ras transfection of RSV-SR transformants leads to suppression of pp6Ov-scr and of H2O2R. In this study we have examined (1) mechanisms of antioxidant activity (AOA) of HEFs transformed by these agents and (2) the possible role of the v-src gene in unusually high AOA of RSV-SR transformants and of activated ras oncogenes in its suppression. All transformants exhibit increased catalase and glutathione peroxidase (GP) activities, while SOD, glutathione and glutathione reductase (GR) were reduced. As compared with other transformants, the significantly higher catalase and the low SOD activities were characteristic of RSV-SR-transformants, while an increase in GP was observed in all types of transformants. Correspondingly, RSV-SR-transformants showed an extremely high H202-catabolizing activity (H2O2CA) and no lipid peroxidation chain reaction (LPCR). N-ras-induced suppression of pp60v-scr of RSV-SR-transformed HEFs coincided with the suppression of catalase, GP, H202 and H202CA. However, suppression of catalase and GP was also observed in N-ras- and Ha-ras-transfected, spontaneously transformed HEFs. Thus, extremely high catalase activity and suppression of LPCR are apparently the main mechanisms of the unusually high H202R of RSV-SR transformants, while its suppression by activated ras oncogenes may also take place in some transformants, free of v-src activity.

Accumulation in "normal" lungs of live tumorigenic cells during sub-cutaneous growth of weakly and highly metastasizing tumors and their in vitro sensitivity to growth regulation by normal fibroblasts.

The main purposes of the study were: (1) in vivo comparison of accumulation of live tumorigenic cells (LTC) in macroscopically normal lungs of animals bearing 6 s.c. Syrian hamster sarcomas differing in spontaneous metastasizing activity (SMA); (2) in vitro examination of the sensitivity of these cell strains to the growth-regulating signals of normal fibroblasts. Cell strains used differed in SMA from very weak (WM) to extremely high (HM). The numbers of LTC doses in "normal" lung tissue of tumor-bearing animals were determined in s.c. transplantation tests by titrating single-cell suspensions prepared from the lung tissues of 5 tumor-bearing animals, for each cell strain, every 5 days during 30 days of s.c. tumor growth, until the appearance of the first spontaneous lung metastases. The sensitivity of WM and HM cells to growth-regulating signals of normal hamster embryo fibroblasts (HEFs) was examined by in vitro co-culturing during 6 days with daily determination of 3H-TdR incorporation in the WM and HM cells grown with or without contact with HEFs. The data presented demonstrate (1) the surprisingly similar efficiency in the occupation of macroscopically normal lung tissues by live tumorigenic cells of WM and HM strains, disseminating spontaneously from the s.c. tumors; (2) the significantly lower sensitivity of HM cells, in contrast to WM, to growth inhibition by contact with HEFs and especially their marked ability to usurp the growth-stimulating signals of normal HEFs.

Allogenic bone-marrow transfusion suppresses development of lung metastases in osteogenic sarcoma patients after radical surgery.

On the basis of experimental data obtained in Syrian hamsters, demonstrating the highly efficient suppression of experimental and spontaneous metastases of highly-metastatic sarcoma cells by the use of allogeneic normal bone-marrow cells (BMC), a clinical protocol for the suppression of lung metastases of osteogenic sarcoma was started in 1984 in the Cancer Research Center, Moscow. From this time onwards, 24 osteogenic sarcoma patients, at stages 2A and 2B were treated with a combination of radical surgery and a single transfusion of normal (non-activated) allogeneic BMC (blood-group and Rhesus compatible). The first results of this ongoing study are now presented. Metastases appeared in 11 out of the 24 patients, generally very early during the first 3-9 months after treatment and in no case after 2 years. More than 50% of the BMC-treated patients were free of lung metastases after 2 or more years of observation; 8 out of 15 are still metastasis-free after 3-4 or more years of observation following treatment. The differences in the frequency of metastasis and duration of survival without metastasis of treated patients compared with a group of 41 osteogenic sarcoma patients at stages 2A and B, treated with radical surgery only (controls) reached significant levels 12 months after treatment and thereafter. Rapid recovery of NK cytotoxic activity has been observed in nearly all successfully BMC-treated patients.

Modulation of pp60v-src and pp60c-src expression in Rous sarcoma virus-transformed hamster fibroblasts transfected with activated N-ras.

Three phenotypically different hamster cell lines transformed with Rous sarcoma virus (RSV) were transfected with plasmid DNA containing an activated N-ras oncogene, and nine clones expressing various levels of p21N-ras were characterized. We examined the effects of p21N-ras on expression and kinase activity of resident src proteins by using a variety of assays that allowed us to discriminate between viral and cellular src proteins. In eight clones with a 10- to 20-fold increase in p21N-ras levels relative to the endogenous protein, we observed a marked reduction in the synthesis and kinase activity of p60v-src. This decrease correlated with transcriptional downregulation of RSV genomic and v-src subgenomic mRNAs. In the same cells, we found a concomitant accumulation of p60c-src and, accordingly, an increase in its protein kinase activity without an apparent increase in c-src mRNA levels. Therefore, modulation of viral and cellular src proteins in cells overexpressing p21N-ras appeared to result from two distinct effects: a downregulation of long terminal repeat-driven transcription and a more complex interaction with cellular effectors that control the stability of p60c-src. Such modulation also seemed to depend on the levels of p21N-ras and, possibly, on host-cell factors, since it was not observed in the third cell line, in which the relative increase in p21N-ras was only 2.5-fold to fivefold.

Clustering of discrete cell properties essential for tumorigenicity and metastasis. III. Dissociation of the properties in N-ras-transfected RSV-SR-transformed cells.

We have previously shown that RSV-SR-transformed hamster cells acquire high resistance to H2O2, i.e. the cytotoxic product of activated macrophages (H2O2R) and that they begin to secrete PGE (PGES), thus inactivating the CTA of NK cells. Among normal cells, the same phenotype is expressed in activated macrophages. In all our RSV-transformed cells these 2 properties were jointly expressed and correlated with high tumorigenicity and experimental metastasizing of these cells. We now show that transfection of 3 RSV-SR-transformed cell strains with activated N-ras leads either to complete inhibition of the H2O2R + PGES phenotype in all clones of one strain, or to inhibition of PGES only in the majority of clones of 2 other strains. Unexpectedly, the complete or partial inhibition of this phenotype did not alter the high tumorigenicity of 2 strains of these cells, but lower tumorigenicity was evident in almost all clones of the third strain (as well as in some gene-neo-transfected clones of these strains). The loss of PGES made these cells susceptible to the CTA of NK cells, while the loss of H2O2R did not alter their resistance to the CTA of macrophages. Expression of the H2O2R + PGES phenotype was retained in all cloned variants of control, gene-neo-transfected cells. The possible relation of the N-ras gene to regulation of src gene activities in RSV-SR-transformed cells is discussed.

Clustering of discrete cell properties essential for tumorigenicity and metastasis. II. Studies of Syrian hamster embryo fibroblasts transformed by Rous sarcoma virus.

Tumorigenic and metastasizing activities (TGA; MA) and susceptibility, or resistance to H2O2 and PGE-releasing activity (H2O2R + PGEs+ phenotype) have been examined in 6 Syrian hamster embryo cell strains transformed in vitro with Rous sarcoma viruses (Schmidt-Ruppin and Prague strains). Early observations of extremely high level of TGA and even MA of RSV-SR-transformants never selected in vivo have been confirmed. The correspondence of these properties with a high level of expression of H2O2R + PGEs+ phenotype and its clustering character were demonstrated in 4 RSV-SR transformants, while significantly lower expression of all these characteristics, including TGA, was observed in 2 RSV-Prague transformants. High level of spontaneous MA was noticed in some RSV-SR transformants. A tumor cell line induced in vivo by RSV-SR did not differ from the cell strain transformed in vitro by RSV-SR. Inhibition of H2O2R + PGEs+ phenotype in one of RSV-SR transformants was obtained with non-toxic doses of BCNU and indomethacin, leading to a marked decrease of TGA.

Clustering of discrete cell properties essential for tumorigenicity and metastasis. I. Studies of Syrian hamster embryo fibroblasts spontaneously transformed in vitro.

The expression of two discrete cell properties related to the host natural effector mechanisms, i.e., resistance to damage by H2O2, a cytotoxic product of activated macrophages, and the ability to secrete PGE, which inhibits NK-cell cytotoxicity, has been examined in parental Syrian hamster embryo cells spontaneously transformed in vitro (STHE strain) and in 18 in vivo selected sublines. In all cell variants, resistance to H2O2 and PGE-releasing activity were either both expressed, or not expressed at all. Parental STHE cells and 5 variants selected in vivo, which were equally highly susceptible to H2O2-induced damage, did not release any detectable amount of PGE upon contact with NK cells. In contrast, 13 other STHE variants selected in vivo and characterized by their resistance to H2O2, all released PGE upon contact with NK cells. Thus, these two biochemically unrelated cell phenotypic characteristics are likely to be either simultaneously selected in vivo, or united in cluster which pre-exist or appear in rare cell variants of the parental cell population in the conditions of in vivo natural selection pressure.

[Differences in the distribution of proviruses in low- and highly- metastatic variants of hamster cells, transformed by Rous sarcoma virus]. / Razlichiia v raspredelenii provirusov v nizko- i vysoko- metastaticheskikh variantakh kletok khomiakov, transformirovannykh virusom sarkomy Rausa.

The structure of provirus in Syrian hamster cells, transformed by Rous sarcoma virus (RSV) varying in metastatic capability in vivo has been analysed. The original cell line and its low metastatic variants contain only one copy of the integrated RSV genome. The DNA of highly metastatic cell lines cloned from the same primary culture, contain an additional copy of provirus. This RSV copy in different highly metastatic variants has a similar integration site.

Natural host resistance and in vivo selection of malignant tumour cells.

In cells transformed in vitro by different agents we have identified two discrete characteristics essential for in vivo selection by the effectors of host natural resistance. These two biochemically distinct characteristics, ie resistance to hydrogen peroxide and ability to secrete E type prostaglandin (PGE) in contact with NK cells, macrophages and neutrophils, may both be expressed either in about 100% of cells transformed by Rous sarcoma virus, or in less than 0.1% of cells transformed spontaneously. The expression of these characteristics provides the cells with selective advantages in vivo and determines the level of their tumorigenic activity and experimental metastasis-forming activity. These characteristics are apparently necessary, but insufficient, for spontaneous metastasizing activity. Demonstrated NK resistance of malignant cell variants is directly connected with their ability to produce PGE. The in vitro transformed cells which do not possess these qualities apparently have no ability for in vivo survival and growth. Though these two cellular properties are connected with biochemically different pathways (catabolism of H2O2 and metabolism of arachidonic acid) they are jointly expressed, or not expressed, in transformed cells. The possible mechanisms of in vivo selection of malignant cell variants, with one or a few cell properties united in clusters, and the dual role of natural resistance in this process are discussed.