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Recent reports demonstrated that neovasculature of certain murine tumours inhibits migration of lymphocytes to malignant tissues. We examined the possible existence of this phenomenon in human prostate adenocarcinoma by relating extent, patterns and composition of leucocyte infiltrates in adenocarcinoma specimens (N=28) to microvessel density and percentages of these vessels expressing adhesion molecules CD54, CD106 and CD62E. Specimens of nodular hyperplasia (N=30) were used as a control for nonmalignant prostate. Increased microvessel density was detected in foci of adenocarcinoma, as compared with adjacent benign areas (P=0.004) or hyperplastic specimens (P=0.001). Only CD54 was detected on prostate vasculature; percentages of CD54-expressing vessels in adenocarcinoma lesions and adjacent areas were higher than in hyperplasia (P=0.041 and P=0.014, respectively). Infiltrating leucocytes were either scattered diffusely in tissue or organised into clusters mainly composed of CD4-positive lymphocytes; smaller percentage of tissue was occupied by clustered infiltrates in adenocarcinoma foci (mean=0.7; median=0; range=0-5) than in adjacent tissue (mean=2.5; median=1; range=0-15; P=.021) and hyperplasia (mean=1.9; median=2; range=0-5; P=.006). In adenocarcinoma foci, microvessel density tended to negatively correlate with percentage of tissue occupied by an overall leucocyte infiltrate (mean=8.6; median=7.5; range=30) and negatively correlated with percentage of tissue occupied by clustered infiltrate (P=0.045). Percentage of CD54-expressing vessels positively correlated with percentage of tissue occupied by an overall (mean=12; median=10; range=30; P=0.01) and clustered (P=0.023) infiltrate in hyperplasia, whereas in carcinoma-adjacent benign areas, correlation was detected only for clustered infiltrates (P=0.02). The results indicate that impaired access of lymphocytes to malignant lesions is associated with increased numbers of newly formed blood vessels, whereas vascular CD54 likely contributes to extravasation of lymphocytes only in benign prostate tissue.

The role of microenvironment interleukin 1 (IL-1) on 3-methylcholanthrene (3-MCA)–induced carcinogenesis was assessed in IL-1–deficient mice, i.e., IL-1β−/−, IL-1α−/−, IL-1α/β−/− (double knockout), and mice deficient in the naturally occurring inhibitor of IL-1, the IL-1 receptor antagonist (IL-1Ra). Tumors developed in all wild-type (WT) mice, whereas in IL-1β–deficient mice, tumors developed slower and only in some of the mice. In IL-1Ra–deficient mice, tumor development was the most rapid. Tumor incidence was similar in WT and IL-1α–deficient mice. Histologic analyses revealed fibrotic structures forming a capsule surrounding droplets of the carcinogen in olive oil, resembling foreign body–like granulomas, which appeared 10 days after injection of 3-MCA and persisted until the development of local tumors. A sparse leukocyte infiltrate was found at the site of carcinogen injection in IL-1β–deficient mice, whereas in IL-1Ra–deficient mice, a dense neutrophilic infiltrate was observed. Treatment of IL-1Ra–deficient mice with recombinant IL-1Ra but not with an inhibitor of tumor necrosis factor abrogated the early leukocytic infiltrate. The late leukocyte infiltrate (day 70), which was dominated by macrophages, was also apparent in WT and IL-1α–deficient mice, but was nearly absent in IL-1β–deficient mice. Fibrosarcoma cell lines, established from 3-MCA–induced tumors from IL-1Ra–deficient mice, were more aggressive and metastatic than lines from WT mice; cell lines from IL-1–deficient mice were the least invasive. These observations show the crucial role of microenvironment-derived IL-1β, rather than IL-1α, in chemical carcinogenesis and in determining the invasive potential of malignant cells. [Cancer Res 2007;67(3):1062–71]

Interleukin-1 (IL-1) comprises a family of closely related genes; the two major agonistic proteins, IL-1alpha and IL-1beta, are pleiotropic and affect mainly inflammation, immunity and haemopoiesis. IL-1beta is active solely in its secreted form, whereas IL-1alpha is active mainly as an intracellular precursor. IL-1 is abundant at tumour sites, where it may affect the process of carcinogenesis, tumour growth and invasiveness and the patterns of tumour-host interactions. Here, we review the effects of micro-environment- and tumour cell-derived IL-1 on malignant processes in experimental tumour models. We propose that membrane-associated IL-1alpha expressed on malignant cells stimulates anti-tumour immunity, while secretable IL-1beta derived from the micro-environment or the malignant cells, activates inflammation that promotes invasiveness and induces tumour-mediated suppression. Inhibition of the function of IL-1 by the inhibitor of IL-1, interleukin-1 receptor antagonist (IL-1Ra), reduces tumour invasiveness and alleviates tumour-mediated suppression, pointing to its feasible use in cancer therapy. Differential manipulation of IL-1alpha and IL-1beta in malignant cells or in the tumour's micro-environment may open new possibilities for using IL-1 in cancer immunotherapy.

We have recently reported that reconstitution of expression of major histocompatibility complex (MHC) class I glycoproteins in MHC-deficient and highly metastatic B16BL6 melanoma cells attenuates their malignant capacities by modulation of compartmentalization and functions of cell membrane receptors for growth factors [Assa-Kunik E, et al. J Immunol 2003;171:2945-52]. Our present study provides evidence that re-expression of an H-2K MHC class I-encoding gene in these cells also augments the expression of the Tap-2 peptide transporter and the inducible proteasome subunits, i.e. Lmp-2, Lmp-7 and Lmp-10. Up-regulation of inducible proteasome subunits was also followed by a significant changed in the proteolytic activity of the proteasome complex. We suggest that, in addition to providing a framework for proper presentation of antigenic peptides, MHC class I glycoproteins may regulate the immune response by modulating the expression and function of other genes, whose products are essential for proper antigen processing and presentation.

Only limited data are available on the early immunological events associated with human cutaneous leishmaniasis (CL). In this study, peripheral-blood mononuclear cells were obtained from 66 individuals (34 patients with cutaneous lesions and 32 apparently healthy controls) who had each spent no more than 3 months in the endemic region of Qetzioth, in southern Israel. These cells' responses to Leishmania major antigen were then explored, by the flow-cytometry-based evaluation of blast transformation (BT). The lymphocytes from 17 (50%) of the patients but only one (3%) of the controls displayed BT. When, in an ELISA, most (52) of the subjects were checked for anti-L. major antibodies, none of the 22 controls investigated but 19 (63%) of the 30 patients were found seropositive. Although 14 (47%) of the 30 patients who were checked for antibodies were BT-positive, the seropositive patients were not significantly more or less likely to be BT-positive than the seronegative patients (P

There is a growing body of evidence that the occurrence of neuroendocrine (NE) differentiation in prostate carcinoma correlates with poor prognosis, tumor progression, and androgen-independence. In the present study, the expression of common NE markers, i.e., chromogranin A (ChGA), serotonin (5HT), neuron-specific enolase (NSE) and adrenomedullin (AM), was retrospectively examined in formalin-fixed, paraffin-embedded prostate tissue samples obtained from patients with adenocarcinoma and from patients with nodular hyperplasia of the prostatic gland (NHPG) (33 and 28, respectively). The statistical analysis of the results (tested the equality of matched pairs of observations using the Wilcoxon matched-pairs signed ranks test) revealed a more prominent expression of ChGA in benign epithelial cells adjacent to adenocarcinomatic lesions (Peri-PAC) than in the adenocarcinoma (PAC) (p = 0.0049). A similar pattern of expression was detected for 5HT (p = 0.000). When comparing the expression of ChGA and 5HT in tissue samples originating in cancer patients with those obtained from NHPG samples, more ChGA and 5HT were expressed in Peri-PAC than in NHPG (p = 0.0004 and 0.002, respectively). The results obtained raise the possibility that adenocarcinoma cells urge some adjacent benign epithelial cells to differentiate into NE cells, which, in turn, may promote tumor growth and invasion.

In a variety of malignancies, the immune-escape phenotype is associated, in part, with the inability of tumor cells to properly present their Ags to CTLs due to a deranged expression of MHC class I glycoproteins. However, these molecules were found to possess broader nonimmune functions, including participation in signal transduction and regulation of proliferation, differentiation, and sensitivity to apoptosis-inducing factors; processes, which are characteristically impaired during malignant transformation. We investigated whether the deranged expression of MHC class I expression by tumor cells could affect proper receptor-mediated signal transduction and accentuate their malignant phenotype. The malignant and H-2K murine MHC class I-deficient B16BL6 melanoma cells were characterized by an attenuated capacity to bind insulin due to the retention of corresponding receptor in intracellular stores. The restoration of H-2K expression in these cells, which abrogated their capacity to form tumors in mice, enhanced membrane translocation of the receptor, presumably, by modulating its glycosylation. The addition of insulin to H-2K-expressing melanoma cells cultured in serum-free conditions precluded apoptotic death by up-regulating the activity of protein kinase B (PKB)/Akt. In contrast, the deficiency for H-2K characteristic to the malignant clones was associated with a constitutive high activity of PKB/Akt, which rendered them resistant to apoptosis, induced by deprivation of serum-derived growth factors. The possibility to correct the regulation of PKB/Akt activity by restoration of H-2K expression in B16BL6 melanoma cells may be considered as an attractive approach for cancer therapy, since an aberrant activation of this enzyme is characteristic to resistant malignancies.

West Nile virus (WNV) is a mosquito-borne disease found most commonly in Africa, West Asia, and the Middle East, where up to 40% of the human population possesses antibodies. It is an emerging disease in the United States. Humans infected with WNV develop a febrile illness that can progress to meningitis or encephalitis. In mice, WNV causes central nervous system infection, paralysis, encephalitis, and death. Currently, no specific therapy or vaccine has been approved for human use. We examined the prophylactic and therapeutic efficacy of pooled human plasma (PP) and intravenous immunoglobulin (IVIG) for treatment of WNV-infected mice. Full protection was achieved when the infected mice were treated with pooled plasma or IVIG obtained from healthy Israeli blood donors that contained WNV-specific antibodies. Similar treatments using PP or IVIG obtained from US blood donors had no protective effect. Recovery of the lethally infected mice was dependent on the dose and time of IVIG administration. These results indicate that antibodies play a major role in protection and recovery from WNV infection and that IVIG can be used as first-line therapy.

Measles virus (MV) persistence in brain cells has broad effects on different cellular functions. We have previously shown that NS20Y clone, originally derived from C1300 neuroblastoma cells, persistently infected with MV (NS20Y/MS), displays constitutively elevated levels of c-fos and PKC mRNAs, implying MV-mediated effects on transcriptional regulation. Nonetheless, the mode by which virus affects the transcriptional machinery still remains obscure. In order to define this phenomenon, we studied the binding properties of major transcription factors (AP-1 and NFkappaB) in NS20Y/MS cells. Using electrophoretic mobility shift approach (EMSA) with the appropriate oligonucleotide probes, we have found that the persistent MV infection does not affect NFkappaB binding, while the AP-1 binding was significantly decreased. Similar inhibition was not observed in NS20Y cells acutely infected with MV. Anti-measles antibody-mediated restriction of viral gene expression restored AP-1 binding, thus suggesting that measles virus proteins may affect the components of the host transcriptional machinery.

A deregulated activity of PKB/Akt (where PKB stands for protein kinase B) renders tumour cells resistant to a variety of apoptosis-inducing stimuli. Elucidation of the mechanisms responsible for this deregulation is of prime importance for the development of novel anti-cancer drugs. Results of the present study demonstrate that the constitutive activity of PKB/Akt in B16BL6 melanoma cells depends on the integrity of cholesterol-enriched membrane microdomains, since the exposure of cells to cholesterol-depleting agents decreases the phosphorylation of this enzyme, with no change in its total protein level. Inhibitors of Hsp90 (heat-shock protein 90) decreased phosphorylation of PKB/Akt with a similar pattern. Dephosphorylation of the enzyme, as a consequence of raft disintegration, could be precluded by inhibition of serine/threonine (but not tyrosine) phosphatases. Our results imply that destabilization of lipid rafts seemingly affects the association of Hsp90 with the respective serine/threonine phosphatases, thereby increasing the accessibility to PKB/Akt to deactivating phosphatases. We have found recently that reconstituted expression of H-2K class I glycoproteins in class I-deficient B16BL6 cells also decreases the phosphorylation of PKB/Akt. Therefore it is possible that raft-associated regulation of this important enzyme involves both H-2K glycoproteins and Hsp90.

To investigate the effect of persistent measles virus infection on signal transduction in cells of neuronal origin, the mouse neuroblastoma cell line NS20Y/MS, which is persistently infected with measles virus, was used. The results demonstrate an approximate 50% increase in total phosphorylation and a similar increase in protein kinase C (PKC) activity. Western blot analysis with anti-total PKC or anti-PKC-alpha antibodies revealed a significant increase in the level of an 80K immunoreactive PKC in NS20Y/MS cells. Following incubation of NS20Y/MS cells with polyclonal anti-measles virus antibodies, which down-regulate the level of measles virus proteins, total and PKC-mediated phosphorylation returned to the basal level of uninfected cells. This effect was reversible and removal of the antibodies resulted in restoration of the high level of total and PKC-mediated phosphorylation. The release of infectious measles virus was strongly inhibited by incubation of NS20Y/MS cells with the PKC inhibitor, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H-7). These results demonstrate that measles virus induces elevation in cellular phosphorylation which is essential for measles virus production.

We have previously shown that noncytotoxic doses of camptothecin (CPT), a topoisomerase I-specific antagonist, inhibit retrovirus replication in acutely and chronically infected cells. To evaluate the efficacy of CPT as an antiretroviral drug in vivo, we injected newborn BALB/c mice with Moloney murine leukemia virus and adult NFS mice with Friend spleen focus-forming virus. The Moloney murine leukemia virus-injected mice developed lymphoma, and the Friend spleen focus-forming virus-injected mice developed erythroleukemia. CPT, administrated together with the virus or 1 or 2 days after virus injection, prevented the onset of the disease in both cases. We showed that repeated CPT treatments increased the effectiveness of the drug when administrated 3 days after virus injection. This ability of CPT to inhibit retrovirus-induced disease in vivo without causing any apparent toxic side effects suggests its application as a legitimate remedy for the treatment of retroviral diseases.

The effects of the H-ras oncogene on fibroblast cell tumorigenicity and immunogenicity was studied in transfectants of the BALB/c 3T3 clone A31 fibroblastoid cell-line. Cells that were transfected with MC29-LTR-H-ras (98/6) or MC29-LTR-v-myc + H-ras (98/4v) and were inoculated into syngeneic BALB/c mice were tumorigenic in 100% and 60% of animals respectively. By contrast, transfectants containing the pSV2neo plasmid alone (98/1) displayed normal characteristics both in vitro and in vivo. Inoculation of mice with mitomycin-C-treated 98/1 or 98/4v cells induced an effective protective immunity to a challenge of live 98/4v cells, and a partial immunity against 98/6 cells. Mitomycin-C-treated 98/6 cells failed to render immunity against a challenge of either 98/6 or 98/4v cells. To correlate immunogenicity and tumorigenicity of the different cell types with cell-surface-antigen expression, we prepared MAbs against 98/4v cells in syngeneic mice. Immunohistochemical and immunoblot analysis revealed that MAbs 102 and 104 recognized 2 protein band of 70 and 45 kDa respectively, which were expressed predominantly in 98/1 and 98/4v cells. A third immunoreactive protein band of 44 kDa that reacted with MAb 6 was expressed at a similar cell-surface density on all cell types. Cell-differentiation-inducing agents, such as DMSO, retinoic acid or sodium butyrate, were all found to induce 98/6 cell flattening and morphological changes toward a normal phenotype that were followed by up-regulation of the 70- and 45-kDa antigens. The results suggest that regulation of expression of the 70- and 45-kDa molecules is affected by H-ras, and that expression of these cell-surface molecules may be relevant to tumor cell immunogenicity.

Interleukin-I (IL-I) is a major immunoregulatory/proinflammatory cytokine which also affects fibroblast proliferation and function and therefore it was of interest to investigate whether its constitutive expression influences the in vivo tumorigenic potential of transformed fibroblastoid cell lines. Here we report on a strong correlation between the constitutive expression of IL-1α and reduced tumorigenicity, using various series of oncogene-transformed NIH/3T3-derived cell lines which produce the cytokine spontaneously or upon gene transfer, following transfection with the IL-1α cDNA. Reduced tumorigenicity of the constitutive IL-1α producing cell lines was manifested either by inability to grow in animals or by regressions of initially growing tumors, within 2 to 3 weeks from cell inoculation. In contrast, mice inoculated with non-IL-1-producing cell lines developed progressive tumors which ultimately killed the animals. Clones obtained from a non-IL-1-producing met-transformed cell line shifted from a progressive to a regressive phenotype, following transfection with an IL-1α-encoding gene, inserted into an appropriate expression vector, resulting in constitutive expression of the cytokine. The effects of constitutive IL-1 expression on tumor development were observed both in histocompatible (NFS/N) and partially allogeneic (BALB/c) mice; however, they were more pronounced in the allogeneic environment. Fibrosarcomas which are non-IL-1 producers induced progressive tumors in both strains of mice at the same growth rate. The differences between the growth characteristics of the fibrosarcomas in histocompatible vs. partially allogeneic mice suggest that IL-1 exerts adjuvant-like effects which increase the immunogenicity of tumor-cell antigens, and they also argue against the possibility that an IL-1-mediated local non-specific inflammatory response is the major effector mechanism of tumor rejection. Indeed, in subsequent studies we shall report on the importance of specific cellular immune responses, especially cytotoxic T lymphocytes (CTLs), in the eradication of constitutive IL-1-producing fibrosarcomas. Thus, our findings may serve as the basis for novel immunotherapy strategies aimed at the induction of IL-1 expression by cells comprising the neoplasm or alternatively by local application of the cytokine in the vicinity of the tumor. © 1992 Wifey-Liss, Inc.

In a previous study we have shown that a single infectious particle of Moloney murine leukaemia virus per cell is sufficient to facilitate chemical carcinogenesis in normal rat kidney cells. When these cells are exposed to the carcinogen after a low number of passages post-infection (p.i.), cell transformation becomes apparent only after many subsequent passages. On the other hand, when exposure is done after a high number of passages p.i., cell transformation can be detected in the treated culture or at the next passage. It is thus evident that whereas the carcinogenic effect is rapid, the viral effect becomes apparent only after a long period of latency. Here we provide evidence that this viral effect requires multiple proviruses and that the long latent period reflects the time needed for a sufficient accumulation of proviruses in some of the cells. This accumulation may result from multiple rounds of superinfection by virions released into the culture medium, although we cannot exclude other mechanisms of provirus amplification. Our data also suggest that this amplification enhances virus production.

In this study we demonstrate a differential transcription of H-2K and H-2D class-I genes in two different tumor cell clones; one is highly metastatic (IE-7) and the other is not metastatic (IC-9), both derived from the same fibrosarcoma, T-10, induced in an (H-2b x H-2k)F1 mouse. The expression of the two parental H-2K alleles is transcriptionally suppressed in both of these clones. In addition the IC-9 clone does not transcribe also the H-2Dk allele. Our data rule out the possibility that this suppression results from enhanced RNA degradation, impaired polyadenylation, DNA rearrangement, or changes in DNA methylation within these genes. Interferons (IFN) are known to enhance MHC expression by acting on a consensus IFN responsive element present in the promoter region of MHC genes. However, IFN-gamma, which is the most potent IFN in this respect, failed to activate the expression of the silent MHC genes in our cells. This finding may reflect a defect within the promoter region of these genes or changes in their chromatin structure.

In the present study, we found a topoisomerase I (topo I) activity in two strains of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) particles. The topo I activity was located in the EIAV cores and differed from the cellular topo I in its ionic requirements and response to ATP, indicating that these were two distinct forms of this enzyme. Topo I activity was removed from the viral lysates and viral cores by anti-topo I antiserum. The only protein recognized by this antiserum was an 11.5 kd protein in HIV lysate and 11 kd in EIAV lysate. We showed that the 11 kd protein recognized by the anti-topo I antiserum is the EIAV p11 nucleocapsid protein. Furthermore, purified topo I protein blocked the binding of the antibodies to the p11 protein and vice versa, purified p11 protein blocked the binding of these antibodies to the cellular topo I. These results suggest that the EIAV p11 nucleocapsid protein and the cellular topo I share similar epitopes.

In previous studies we have demonstrated that metastatic cells, derived from T-10 fibrosarcoma, express an immunogenically abnormal H-2Dk glycoprotein which is involved in manifesting their metastatic phenotype. In the present study we show that these cells contain a remarkably high level of H-2Dk specific mRNA. Moreover, by cloning cDNA of this gene and analyzing its nucleotide sequence, we found 4 single nucleotide changes. Two of them did not change the encoded amino acids, whereas the others resulted in two amino acid substitutions in the alpha-2 domain of the protein product that might account for its immunogenic abnormality.

We demonstrate in this study that infection with Moloney murine leukemia virus (M-MLV) and exposure to 3-methylcholanthrene (3-MC) can cooperate to transform NIH/3T3 mouse fibroblasts. M-MLV seems to stimulate the expression of c-myc and of a certain major histocompatibility complex (MHC) class I gene. Yet M-MLV infection by itself is insufficient to transform these cells. However, exposure of the infected cells to 3-MC resulted in a rapid cell transformation with concomitant enhancement of c-Ha-ras and H-2K class I MHC gene expression in the transformed cells. No such transformation was observed when uninfected NIH/3T3 cells were similarly treated with this carcinogen. Clones of cells transformed by this combined effect of M-MLV and 3-MC were found to be highly tumorigenic in fully immunocompetent allogeneic BALB/c mice. We provide evidence to suggest that the enhanced expression of the H-2K gene in these transformed cells plays an important role in overcoming the BALB/c allogeneic barrier and allowing tumor growth in these mice.

The regulation of topoisomerase I (topo I) activity is of prime importance for gene expression. It participates in DNA replication, transcription, recombination, and DNA repair, and serves as a target for anticancer drugs. Many proteins and enzymes are modified by O-linked beta-N-acetylglucosamine (O-GlcNAc), which exerts profound effects on their function. However, the modification of topo I by O-GlcNAc and the effect on its activity has not been previously reported. Here, we show that topo I protein is modified by O-GlcNAc in vitro in the porcine proximal tubular epithelial cell line (LLPCK-1), and in vivo in the mouse kidney. The level of O-GlcNAcylation of topo I protein correlates well with the enzyme activity, namely, a decrease in O-GlcNAc results in a reduction in topo I activity, and vice versa. O-GlcNAc transferase (OGT) was coprecipitated with topo I protein, suggesting a possible interaction between both enzymes. In addition, treatment of cells with glucosamine increased topo I activity and O-GlcNAcylation. The results of this study provide a novel mechanism for the regulation of topo I activity. Topo I is important for DNA transcription, therefore, its regulation by GlcNAcylation contributes to the mechanism by which glucose levels affect gene expression, and may pave the way to the development of new drugs that could control topo I activity.

Cancerous ovarian tissues contain and produce high levels of pro-inflammatory cytokines (IL-1, IL-6 and TNF-alpha). The aim of this study was to assess the mechanisms by which autocrine IL-6 affects the ovarian carcinoma continuous cell line (SKOV-3) tumorigenicity.Autocrine IL-6 was neutralized by the addition of anti-IL-6 antibodies to SKOV-3 cell cultures. The proliferation rate was evaluated by MMT staining and the capacity to produce matrix metalloproteinases (MMPs) 2 and 9 was examined by zymography.The SKOV-3 cells secreted IL-6 in a time-dependent manner (24-96 h). The addition of anti-IL-6 antibodies to SKOV-3 cell cultures did not affect their proliferation rate within 96 h of incubation. In addition, SKOV-3 cells secreted MMP-2 and MMP-9 as confirmed by zymography. The MMP-9 levels decreased in a time-dependent manner (3, 8, 24 h) and the addition of anti-IL-6 antibodies to SKOV-3 cell cultures significantly decreased their capacity to secrete MMP-9, particularly after 8 h of incubation. MMP-2 (pro-active and active forms) was also secreted by SKOV-3 cell cultures but could be measured only after 24-96 h of incubation. The levels of MMP-2 increased in a time-dependent manner. The addition of anti-IL-6 antibodies to SKOV-3 cell cultures did not affect their capacity to secrete MMP-2.Our results suggest that IL-6 secreted by SKOV-3 cells could be involved in their tumorigenic potential, particularly potentiating their capacity to secrete MMP-9.

Constitutive expression of cell-associated, but not secreted, interleukin-1α (IL-1α) by oncogene-transformed fibrosarcoma cells induced regressing tumors in mice, a phenomenon that was abrogated by the IL-1 inhibitor, the IL-1 receptor antagonist (IL-1Ra). On the contrary, non-IL-1α-expressing tumor cells induce progressive tumors in mice. In vivo and ex vivo experiments have shown that regression of IL-1α-positive fibrosarcoma cells depends on CD8+ T cells, which can also be activated in CD4+ T cell-depleted mice, with some contribution of natural killer cells. In spleens of mice bearing the non-IL-1α-expressing fibrosarcoma cells, some early and transient manifestations of antitumor-specific immunity, such as activation of specific proliferating T cells, are evident; however, no development of cytolytic T lymphocytes or other antitumor protective cells could be detected. In spleens of mice bearing the non-IL-1α-expressing fibrosarcoma cells, the development of early tumor-mediated suppression was observed, and in spleens of mice injected with IL-1α-positive fibrosarcoma cells, protective immunity developed in parallel to tumor regression. Treatment of mice bearing violent fibrosarcoma tumors with syngeneic-inactivated, IL-1α-positive fibrosarcoma cells, at a critical interval after injection of the malignant cells (Days 5–12), induced tumor regression, possibly by potentiating and amplifying transient antitumor cell immune responses or by ablation of tumor-mediated suppression. Membrane-associated IL-1α may thus serve as an adhesion molecule, which allows efficient cell-to-cell interactions between the malignant and immune effector cells that bear IL-1Rs and function as a focused cytokine with adjuvant activities at nontoxic, low levels of expression. Our results also point to the potential of using antitumor immunotherapeutic approaches using cell-associated IL-1α.

Tumor cells secreting IL-1β are invasive and metastatic, more than the parental line or control mock-transfected cells, and concomitantly induce in mice general immune suppression of T cell responses. Suppression strongly correlates with accumulation in the peripheral blood and spleen of CD11b+/Gr-1+ immature myeloid cells and hematological alterations, such as splenomegaly, leukocytosis, and anemia. Resection of large tumors of IL-1β-secreting cells restored immune reactivity and hematological alterations within 7–10 days. Treatment of tumor-bearing mice with the physiological inhibitor of IL-1, the IL-1R antagonist, reduced tumor growth and attenuated the hematological alterations. Depletion of CD11b+/Gr-1+ immature myeloid cells from splenocytes of tumor-bearing mice abrogated suppression. Despite tumor-mediated suppression, resection of large tumors of IL-1β-secreting cells, followed by a challenge with the wild-type parental cells, induced resistance in mice; protection was not observed in mice bearing tumors of mock-transfected fibrosarcoma cells. Altogether, we show in this study that tumor-derived IL-1β, in addition to its proinflammatory effects on tumor invasiveness, induces in the host hematological alterations and tumor-mediated suppression. Furthermore, the antitumor effectiveness of the IL-1R antagonist was also shown to encompass restoration of hematological alterations, in addition to its favorable effects on tumor invasiveness and angiogenesis that have previously been described by us.

We have previously reported that the capacity of highly malignant B16BL6 murine melanoma cells to induce cell death in naive syngeneic lymphocytes stems from the absence of major histocompatibility complex (MHC) class I glycoproteins in these melanoma cells. Our present study provides evidence that the above-mentioned lymphocidic activities of B16BL6 cells are selectively attenuated when the expression of H-2K (but not H-2D or H-2L) MHC class I glycoproteins is reconstituted in these cells. The induction of apoptosis in naive lymphocytes by H-2K-deficient melanoma cells does not involve the Fas ligand (Fas-L)/FAS signaling module, as demonstrated by employing lymphocytes derived from Fas-L(gld)- or Fas(lpr)-deficient mice in co-culture experiments. Furthermore, these tumor cells fail to induce Fas-L-mediated fratricide in co-cultured lymphocytes and do not express Fas-L either when grown alone or co-cultured with lymphocytes. These findings explain the previously widely reported selective down-regulation of certain MHC class I-encoded glycoproteins (H-2K, bur not H-2D or H-2L) during tumor progression. Namely, the initiation of an effective immune response against H-2K-deficient cells could be abrogated at very early steps, as the result of the induction of Fas-L/Fas-independent cell death among naive lymphoid cells.

In a variety of malignancies, the immune-escape phenotype is associated, in part, with the inability of tumor cells to properly present their Ags to CTLs due to a deranged expression of MHC class I glycoproteins. However, these molecules were found to possess broader nonimmune functions, including participation in signal transduction and regulation of proliferation, differentiation, and sensitivity to apoptosis-inducing factors; processes, which are characteristically impaired during malignant transformation. We investigated whether the deranged expression of MHC class I expression by tumor cells could affect proper receptor-mediated signal transduction and accentuate their malignant phenotype. The malignant and H-2K murine MHC class I-deficient B16BL6 melanoma cells were characterized by an attenuated capacity to bind insulin due to the retention of corresponding receptor in intracellular stores. The restoration of H-2K expression in these cells, which abrogated their capacity to form tumors in mice, enhanced membrane translocation of the receptor, presumably, by modulating its glycosylation. The addition of insulin to H-2K-expressing melanoma cells cultured in serum-free conditions precluded apoptotic death by up-regulating the activity of protein kinase B (PKB)/Akt. In contrast, the deficiency for H-2K characteristic to the malignant clones was associated with a constitutive high activity of PKB/Akt, which rendered them resistant to apoptosis, induced by deprivation of serum-derived growth factors. The possibility to correct the regulation of PKB/Akt activity by restoration of H-2K expression in B16BL6 melanoma cells may be considered as an attractive approach for cancer therapy, since an aberrant activation of this enzyme is characteristic to resistant malignancies.

OBJECTIVES: Reassessment of predisposing factors to transverse lie presentation. METHODS: Retrospective analysis of delivery data on 92 women with transverse lie presentation, using ultrasonography, and 92 randomly chosen control vertex deliveries. RESULTS: The association between transverse lie and multiparity, prematurity, placenta previa, polyhydramnios and uterine anomalies is reaffirmed. Predisposing factors were found in 66% of primiparas but in only 33% of multiparas. CONCLUSIONS: Known predisposing factors to transverse lie presentation withstood a closer and more accurate assessment of their incidence.

The different physiological roles of interleukin-1alpha (IL-1alpha) and interleukin-1beta (IL-1beta) are not well understood, especially when considering the apparent overlap and redundancy of the two IL-1 molecules. Characterization of IL-1alpha and IL-1beta expression was performed in this study in organs from young and old mice, using immunohistochemistry and ELISA (enzyme-linked immunosorbent assay). The results indicate that organ IL-1alpha and IL-1beta display different patterns of expression: IL-1alpha is manifested more prominently in lymphoreticular organs (lungs, small intestine, spleen, liver), while IL-1beta is more evident in highly specialized and more vulnerable organs, which do not play a leading role in defense against infections and intoxication (heart, brain, skeletal muscle, kidney). This differential expression is more accentuated in old mice, possibly pointing to the special relevance of these cytokines to organ homeostasis in old age. These findings may shed new light on the physiological functions of IL-1alpha and IL-1beta, and may also lead to the development of improved therapeutic approaches, based on the specific manipulation of these cytokines.

Recombinant activated factor VII (rFVIIa, NovoSeven) was used in three patients with massive obstetric hemorrhage due to placenta previa accreta, rupture of the uterus and pre-eclampsia with HELLP. Administration of the drug markedly decreased the bleeding and enabled control of the hemorrhage. rFVIIa seems to be an adjunctive hemostatic measure for the treatment of severe obstetric hemorrhage.

The findings presented in this work show that IL-1α and IL-1β display differential and even inverse patterns of organ expression, demonstrated most clearly at old age and beyond the realm of immunology. Based on these IL- 1α /IL- 1β specific patterns of organ expression and considering the distinct physiological function of various organs, IL-1 a may be highlighted more as a potent anti-infectious/toxic agent, which can initiate and potentiate prolonged inflammatory responses, apparently mainly based on activation of cellular immunity. In contrast, it possibly appears that IL-1α may be involved in more restricted inflammatory responses, than conceived before. Other homeostatic functions of both IL-1 molecules can also be envisioned, including for example control of the cell cycle and apoptosis. In this context, diverse and even opposing biological effects of IL-la/IL-lb may be anticipated. The “net” activity of IL-1 in a certain organ may depend on the type of expressed IL-1 and IL-lreceptors, as well as on the locally operant cytokine network. The in vivo interactions between the two IL-1 forms may be of crucial importance to organ homeostasis, especially under the stressful conditions, characterizing old age. Further studies are needed to clarify the full perspective of IL-la and IL-lb roles in organ homeostasis, the understanding and manipulation of which may hopefully lead to development of beneficial therapeutic interventions.

Recent studies have shown that high insulin-like growth factor I (IGF-I) blood level is a risk factor in breast and prostate cancer. The aim of this study was to determine whether the mitogenic activity of IGF-I in mammary cancer cells can be reduced by the dietary carotenoid lycopene. The anticancer activity of lycopene, the major tomato carotenoid, has been suggested by in vitro, in vivo, and epidemiological studies. Growth stimulation of MCF7 mammary cancer cells by IGF-I was markedly reduced by physiological concentrations of lycopene. The inhibitory effects of lycopene on MCF7 cell growth were not accompanied by apoptotic or necrotic cell death, as determined by annexin V binding to plasma membrane and propidium iodide staining of nuclei in unfixed cells. Lycopene treatment markedly reduced the IGF-I stimulation of tyrosine phosphorylation of insulin receptor substrate 1 and binding capacity of the AP-1 transcription complex. These effects were not associated with changes in the number or affinity of IGF-I receptors, but with an increase in membrane-associated IGF-binding proteins, which were previously shown in different cancer cells to negatively regulate IGF-I receptor activation. The inhibitory effect of lycopene on IGF signaling was associated with suppression of IGF-stimulated cell cycle progression of serum-starved, synchronized cells. Moreover, in cells synchronized by mimosine treatment, lycopene delayed cell cycle progression after release from the mimosine block. Collectively, the above data suggest that the inhibitory effects of lycopene on MCF7 cell growth are not due to the toxicity of the carotenoid but, rather, to interference in IGF-I receptor signaling and cell cycle progression.

The specific expression of pro-inflammatory cytokines may affect the functioning of organs in different ways. The results of specific cytokine bioassays used in this study show a distinct pattern of tissue expression of IL-1 IL-6 and CSF-1. Cytokine activity was assessed in conditioned media (CM) and lysates (LYS), obtained from different organs of control or lipopolysaccharides (LPS)-injected mice; LPS representing a potent inflammatory stimulus. Low constitutive levels of IL-1 could be demonstrated only in CM/LYS from organs with lymphoreticular function, such as the liver, spleen, intestine and lungs. On the other hand, IL-6 and CSF-1 were mainly detected in the CM (and not in lysates) of organs, such as the heart, kidneys, muscle and brain. LPS injection basically resulted in an accentuated form of the constitutive pattern. CSF-1 displays a similar pattern of expression to that of IL-6, best detected in CM after LPS stimulation. Thus, a mirror-image relationship emerges between the patterns of IL-1 and IL-6/CSF-1 expression in two groups of organs: those with lymphoreticular function, which manifest high IL-1 and low IL-6/CSF-1 activity, as compared to organs characterized by highly specialized and potentially vulnerable functions (such as the heart, brain, muscle and kidney), which exhibit high IL-6/CSF-1 and low IL-1 activity. Due to their defensive functions, lymphoreticular organs, which are in charge of the 'gates of entry' to the body, mount extensive IL-1-mediated inflammatory responses, even at the cost of possible tissue-damage. On the other hand, the more vulnerable internal organs mount IL-6/CSF-1-mediated responses which are milder and bear less potential for tissue damage. The distinct patterns of expression of pro-inflammatory cytokines in different organs, at steady state or under inflammatory conditions, may shed light on tissue characteristic homeostatic and defence mechanisms.

Objective: To characterize the 24-h arterial pressure (AP) profile and the left ventricular (LV) structures and functions in women with pregnancy-associated hypertension. Methods: Twenty nulliparous pregnant women after 20 weeks' gestation, 10 normotensive and 10 hypertensive women matched for gestational age, were hemodynamically investigated using 24-h AP monitoring and Doppler echocardiography to determine LV structures and functions, both systolic and diastolic. Results: The hypertensive women had significantly higher AP determinations throughout the 24 h, with no change in diurnal variation, i.e. nocturnal decline and early morning peaks. Their LV mass was greater and it was accompanied by a slight reduction in contractility and a significant reduction in LV relaxation. The increased AP was due to peripheral vasoconstriction, while cardiac output was preserved. Conclusions: It appears that pregnancy-associated hypertension is caused mainly by arterial vasoconstriction and not by higher cardiac output. The hypertension increases the LV mass, which is associated with a fall in LV relaxation.

A direct correlation between the constitutive expression of IL-1α and reduced tumorigenicity of fibrosarcomas was observed. This was established in fibrosarcoma cell lines which produce IL-1α ‘spontaneously’, possibly as an aberration of oncogene-mediated transformation or upon IL-1α gene transfer. In fibroblasts intracellular or membrane-associated IL-1α is expressed, whereas the secreted form of the cytokine (IL-1β) is absent. Studies on the mechanisms of tumor regression of the IL-1α-positive fibroblastoid cell lines indicated that IL-1α potentiates the development of tumor cell-specific CTLs, which are of importance for tumor eradication. Thus, IL-1α induces enhanced helper T cell activity which provides auxiliary signals for the growth/development of CTLs. Non-adaptive effector cells, activated locally by IL-1α-expressing fibrosarcoma cells, also contribute to the eradication of IL-1α-expressing fibrosarcomas. Local IL-1α expression potentiated antigen presentation, by the malignant fibroblasts as well as by tissue-resident antigen-presenting cells, thus further potentiating anti-tumor immune responses. Mice, in which IL-1α-producing tumors were regressed, developed an immune memory and rejected a challenge with an IL-1 non-producing violent tumor cell line. Endogenous IL-1α activates a cytokine cascade (i.e.; IL-6, CSF), produced by the malignant cells and possibly also by stromal cells. However, IL-1α expression is essential for fibrosarcoma eradication, while other cytokines possibly amplify and sustain its action. IL-1 non-producing fibrosarcomas can be induced to express IL-1α in a transient manner, by treatment in culture with cytokine/LPS, and subsequently they shift from progressor to regressor tumors. Positive immunotherapeutical effects were obtained when treating mice bearing IL-1 non-producing fibrosarcomas with cells from the same line, induced to express IL-1α in a constitutive or transient manner. Thus, IL-1α expression by malignant cells bears the potential to be applied in immunotherapy, as it is assumed that most naturally occurring tumors do not express IL-1α activity. Further studies are aimed at the elucidation of the mechanisms of IL-1α-mediated tumor regression and on the development of immunotherapy protocols based on its expression by malignant cells.

In an attempt to define immunological parameters affected by the H- ras oncogene, we have used Balb/c 3T3 cells transfected with either H- ras ( 98 6 ), H- ras + v- myc ( 98 4 v ) or plasmid only ( 98 1 ). We found that while control and oncogene-transfected Balb/c 3T3 cells exhibit similar low sensitivity to lysis by natural killer (NK) cells, H- ras + v- myc -transfected cells could immunize syngeneic Balb/c mice and induce cytotoxic T cells (CTL) with broad specificity, that lysed all types of Balb/c 3T3 cells tested. Immunization of Balb/c mice with 98 4 v cells prevented homologous tumor formation and partially inhibited the formation of tumors derived from H- ras -transfected cells. 98 6 cells were not immunogenic in vivo and did not protect the animals from a challenge of 98 6 cells. The results suggested that CTLs but not NK effector cells were important for eliciting in vivo tumor rejection of H- ras + v- myc -transfected cells. In contrast, antigens eliciting the cytotoxic T-cell response, and possibly also the in vivo tumor cell rejection response, were expressed on all cell types tested but were immunogenic only on the surface of 98 4 v cells. We further determined major histocompatibility complex (MHC) class-I molecule expression on the outer cell surface and found that H-2K was down-regulated in H- ras -transfected cells. The results support the observation that oncogenes can down-regulate specific MHC antigens, thereby preventing presentation of tumor antigens and allowing tumor escape from immune recognition.

In this present study we have characterized the array of hemopoietic cytokines generated by fibroblasts in response to inflammatory signals. It was shown that murine embryo fibroblasts (MEF) are able to generate colony stimulating factors (CSFs) [granulocyte-macrophage (GM-CSF), macrophage-CSF (CSF-1) and granulocyte-CSF (G-CSF)] as well as the hemopoietin interleukin 6 (IL-6), while the production of IL-3, IL-4 or tumor necrosis factor (TNF) could not be detected in MEF, as assessed by bioassays or expression of specific mRNA. The production of colony promoting activity was observed when fibroblasts were stimulated by lipopolysaccharide (LPS) or individual cytokines [IL-1, interferon-γ (IFN-γ), IL-2, IL-4 or TNF] in serum-free conditions as well as by serum itself. These inducers differentially stimulated in MEF the production of various CSFs; LPS induced mainly CSF-1, while cytokines or serum induced equivalent amounts of GM-CSF and CSF-1. The production of IL-6 was induced by LPS in serum-free conditions, while stimulation by cytokines (IL-1 or IFN-γ) resulted in IL-6 production only in serum-supplemented cultures. Serum by itself did not induce IL-6 production by MEF. The secretion of IL-6 by fibroblasts was detected early and peacked after 6 hours, while CSF activity peacked after 24–72 hours, depending on the inducer. Constitutive mRNA expression of CSF-1 was detected in serum-free conditions in unstimulated MEF, however colony-promoting activity was detected only upon stimulation with cytokines, LPS or serum. Transcription of specific mRNA without detectable biological activity was also observed for IL-6 in MEF stimulated with IL-1 in serum-free conditions. Thus, multiple control levels are responsible for the production of hemopoitic cytokines by fibroblasts that may result in the amplification and fine tuning of immune/inflammatory reactions and hemopoietic responses.

In the present study we investigated the effect of temperature on MHC class-I gene expression in BALB/C 3T3 cells incubated for 5 days at 34 degrees C, 37 degrees C and 39 degrees C. FACS analysis revealed no significant difference in the cell surface expression of any of the 3 major class-I antigens at 34 degrees C and 37 degrees C. Strikingly, however, when the level of the respective mRNA was determined, only that of the H-2K was comparable at both temperatures, whereas the levels of the H-2D and H-2L mRNA were profoundly higher at 37 degrees C. These data appear to reflect a differential temperature-related transcriptional control of the different class-I genes or a different temperature effect on the stability of their mRNA. The absence of a parallel increase in surface expression of the corresponding H-2D and H-2L antigens may result from some translational or post-translational limiting factors. At 39 degrees C, however, these limiting factors seem to be overcome since the surface expression of all the 3 antigens was remarkably increased although the level of their encoding mRNA was rather lower than in 37 degrees C. This stimulatory effect might be ascribed to heat shock proteins which are known to arise in cells at heat or other stress conditions. They participate in assembly and disassembly of various protein complexes and in transport of certain proteins across intracellular membranes. Such proteins may have arisen in our cells at 39 degrees C and facilitated the intracellular assembly of the class-I molecules and their transport to the cell surface. The possible implication of such heat shock proteins in the anti-tumor effect of hyperthermia is discussed.

Oncogene-transformed BALB/c-3T3 fibroblasts which spontaneously or upon immune-activation with cytokines and lipopolysaccharide (LPS) generate IL-1 alpha, were tested for their tumorigenicity as well as their interaction with natural immune defense by NK cells and macrophages. Oncogene-transformed fibroblasts were weakly tumorigenic, since not all mice developed tumors despite application of high doses of tumor cells. This was independent of the immune status of the host. However, in the immunocompetent host those transformed fibroblast lines which spontaneously produced IL-1 alpha grew only transiently and then regressed. After induction of IL-1 alpha production, a decrease in the rate of tumor take was noted and the rate of regression of developing tumors was increased. Regression of IL-1-producing transformed fibroblasts was strongly reduced but not completely abolished in sublethally irradiated mice. This indicated that IL-1 production may predominantly influence T-cell-mediated defense, but some influence on non-adaptive immunity could not be excluded a priori. IL-1 production did not influence susceptibility of transformed fibroblasts towards NK cells and macrophages. However, IL-1-producing transformed fibroblasts were most potent stimulators of NK cells and macrophages, the stimulatory effect being locally restricted. In conclusion, IL-1 producing, oncogene-transformed fibroblasts which generated the cytokine constitutively or upon immune-activation, were rejected from the tumor-bearing host following initial growth. Fibroblast-induced local activation of NK cells and macrophages was shown to play some role in tumor graft rejection. The influence of IL-1 production of transformed fibroblasts on T-cell-mediated defense is addressed in the accompanying report.

Oncogene-transformed fibroblasts which expressed IL-1, spontaneously or after activation with conditioned medium (CM) and lipopolysaccharide (LPS), regressed in the syngeneic host. Since regression was significantly influenced by the immune competence of the host (see companion report), we speculated that regression was T-cell-mediated. Frequencies of cytotoxic T-cell precursors (CTLp) were in the same range for activated and non-activated, transformed fibroblasts. Furthermore, it was found that lysability of transformed fibroblasts was not influenced by expression of IL-1. These findings exclude the possibility that regression of CM- and LPS-treated transformed fibroblasts may have been due to the appearance of new, strongly immunogenic epitopes. On the other hand, frequencies of CTL were significantly increased after in vivo immunization with IL-1-expressing as compared to IL-1-non-expressing transformed fibroblasts. The in vivo maturation/expansion of CTL could have been the consequence of activation of helper T cells (TH), transformed fibroblast-associated IL-1 delivering the co- stimulatory signal. Analysis of frequencies and proliferation rates of TH confirmed this assumption. Both parameters were significantly increased after stimulation with transformed fibroblasts expressing IL-1, in comparison to transformed fibroblasts not expressing IL-1. Furthermore, purified T cells apparently depleted of cells expressing MHC class-II antigens, i.e. antigen- presenting cells, proliferated in the presence of transformed fibroblasts expressing IL-1. Since IL-1 rather than MHC class-II antigen expression was the limiting factor, antigen presentation by IL-1-expressing transformed fibroblasts appears unlikely. Instead, maturation of antigen-presenting cells could well have been initiated by tumor-associated IL-1. We conclude that IL-1 expression of transformed fibroblasts plays an important role in the induction of a T-cell-mediated anti-tumor response. The effect is due to increased efficiency in the activation of helper T cells and may be supported by activation of antigen-presenting cells.

I. Levin, B. Klein, S. Segal, A. Eyal, J. Gopas, J. Kaneti, M. Saadon, O. Kuperman. Expression of HLA class I-encoded cell surface antigens in transitional cell carcinoma of the urinary bladder.

We present 3 patients with papillary renal adenocarcinoma who were initially misdiagnosed owing to unusual clinical presentation. We suggest that if these presentations are borne in mind, pre-operative detection of this uncommon condition should be possible more often than is now the case.

We have introduced point mutations in v-rasH to study their effects on biochemical and biological properties of the ras-encoded protein p21. Several of these mutant proteins do not bind GTP and thus lack GTPase activity, while others were shown to have their GTP binding reduced. We have introduced these ras mutants into NIH 3T3 fibroblastoid cells to study major parameters of clinical importance which are associated with neoplastic transformation, particularly MHC expression in cells, metastasis and tumorigenesis in both nude mice and immune competent mice. Our data show that certain mutations in v-ras differentially affect the expression of the transformed phenotype. Mutant ras molecules deficient in GTP binding fail to generate rapidly progressing tumors in immune competent mice, and not all morphologically transformed cells were capable of experimental metastasis. Cells transformed by certain v-ras mutants form tumors in immunocompetent mice and show reduced expression of MHC class-1 antigens. Other cells are morphologically transformed and tumorigenic in athymic nude mice, but fail to form tumors in normal mice and show levels of MHC class-1 antigen expression similar to non-transformed 3T3 cells. The inverse relationship between MHC class-1-antigen expression and the degree of transformation in fibroblastoid cells suggests that the ras gene product could be involved in regulating MHC expression.

In the present study we have demonstrated that fibroblasts can generate the inflammatory cytokine interleukin 1 (IL 1) under conditions similar to those abundant in cellular immune responses. Thus, induction of IL 1 requires a sequential two-step protocol which consists of preactivation of mouse embryo fibroblasts (MEF) with crude preparations of T cell or macrophage-derived conditioned media (CM; 72 h), followed by a challenge with lipopolysaccharide (LPS; 24 h). Unstimulated fibroblasts or such cells activated by either CM or LPS produced only low levels of IL 1, while a synergism between both signals was observed for obtaining maximal IL 1-like activity in MEF. Each of a series of individual recombinant lymphokines and cytokines (IL 2, granulocyte/macrophage-colony-stimulating factor, tumor necrosis factor, IL 1 beta and interferons-alpha, beta and gamma) was shown to serve as an efficient priming signal for the induction of IL 1. IL 1-like activity in fibroblasts was detected in cell lysates or associated with the producing-cell membrane but not in culture fluids. Immune-stimulated fibroblasts, activated under such experimental conditions, were shown to actively transcribe mRNA of both IL 1 genes (alpha and beta). For the expression of IL 1-specific mRNA in fibroblasts a single stimulus, provided by either LPS or a lymphokine/cytokine, was sufficient; however, a more intense signal was observed when both stimuli were applied. The IL 1-like biological activity of fibroblast origin was significantly reduced by anti-IL 1 alpha antibodies. Thus, fibroblasts, when activated by immune and bacterial products, generate IL 1 which in turn possibly amplifies cellular immune responses or inflammatory processes in connective tissues.

The Ras family is the most widely mutated group of human proto-oncogene. Previously, we found an organic Tellurium compound, AS101, which exhibits immunomodulatory activity and strong anti-tumor effects. Whether these properties can be exploited for cancer therapy was examined using v-Ha-Ras transformed and V-mos transformed fibroblasts cells. In this study AS101 appeared to selectively block ~90 % of the proliferative growth of v-Ha-Ras transformed cells, while not affecting the growth of V-mos transformed cells. Furthermore, the proliferative growth inhibiting effect of AS101 was prevented when the activity of p21Ras was blocked by expression of a dominant negative mutant 116y of the v-Ha-Ras or by treating Ha-Ras transformed cells with a farnesyltransferase inhibitor. The anti-proliferative effect of AS101 was accompanied by morphological changes typical of apoptosis i.e.: membrane blebbing, condensation of nuclear chromatin, and formation of apoptotic bodies. AS101-induced apoptosis of v-Ha-Ras transformed cells correlated with suppression of Akt protein expression, activation of caspases, and induction of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Thus, our results suggest that AS101 can be used to effectively treat tumors which, are induced due to an oncogenic mutation in p21Ras encoding gene and to selectively block oncogenic function of mutated Ha-Ras in human tumors.

Two clones of the 3LL Lewis lung carcinoma, a low-metastatic clone A9 and a high-metastatic clone D122, were studied for MHC expression and immunogenic properties. Using monoclonal antibodies, we demonstrated that the A9 clone expresses both the H-2Kb and the H-2Db, whereas the D122 expresses only the H-2Db, and lacks the expression of the H-2Kb encoded molecules. Cells of the low-metastatic clone A9 grew progressively in syngeneic (C57BL/6J) or in F1 mice, but were rejected in allogeneic recipients. The high-metastatic D122 grew progressively in all mouse strains tested, yet metastases were formed only in syngeneic recipients. When H-2 recombinant mice were used, the A9 again manifested a significantly greater immunogenic potency than the metastatic D122, which grew in all 4 recombinants tested. Metastases, however, were formed in B10HTG and to a lesser extent in B10A(4R), thus indicating that metastasis formation is restricted by both C57BL background and H-2Db sub region. We subsequently tested whether the higher immunogenicity of the H-2Kb-positive A9 cells is expressed also in syngeneic mice, to examine whether this could account for its low metastatic phenotype. We found that immunization by A9 cells significantly inhibited the growth of a subsequent A9 graft and even of D122, yet D122 did not retard the growth of secondary D122 or A9 cells. The increased immunogenic effect was expressed also in the generation of syngeneic cytotoxic lymphocytes by A9 but not by D122 cells. We suggest that expression of H-2K molecules on the 3LL clones, immunogenicity and the metastatic phenotype are causally related in this system.

We have used the 3-Methylcholanthrene induced T-10 fibrosarcoma tumour cell system (H-2b xH-2k)F1 to elucidate the possible correlation between metastatic potential, expression of individual H-2 antigens and susceptibility to NK cells. Transfection of the non-metastatic and NK sensitive IC9 cells (Db+, Dk-, Kb-, Kk-) with the H-2Dk gene, altered the metastatic phenotype of the parental cells, yet had no effect on the susceptibility of these tumour cells to lysis by NK and did not elicit a specific CTL response in syngeneic hosts. Variants of the metastatic and NK resistant IE7 clone (Db+, Dk+, Kb-, Kk-), lacking H-2Dk, were selected by treatment with monoclonal anti H-2Dk antibodies and complement. These variants were sensitive to NK and poorly or non metastatic. Retransfection of 'Dk' 'loss' variants with the H-2Dk gene, resulted in the isolation of several clones expressing a wide range of metastatic phenotypes but maintained sensitivity to NK. These results indicate that the H-2D region of the MHC and or closely linked genes may be involved in the complex interrelationship between target susceptibility to NK and metastasis.