Shraga Segal

LYCOPENE INTERFERES WITH CELL CYCLE PROGRESSION AND INSULIN-LIKE GROWTH FACTOR I SIGNALING IN MAMMARY CANCER CELLS

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.

CD11b+/Gr-1+ Immature Myeloid Cells Mediate Suppression of T Cells in Mice Bearing Tumors of IL-1β-Secreting Cells

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.

Prophylactic and Therapeutic Efficacy of Human Intravenous Immunoglobulin in Treating West Nile Virus Infection in Mice

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.

Interleukin-1β–Driven Inflammation Promotes the Development and Invasiveness of Chemical Carcinogen–Induced Tumors

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-1beta(-/-), IL-1alpha(-/-), IL-1alpha/beta(-/-) (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-1beta-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-1alpha-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-1beta-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-1alpha-deficient mice, but was nearly absent in IL-1beta-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-1beta, rather than IL-1alpha, in chemical carcinogenesis and in determining the invasive potential of malignant cells.

Differential effects of IL-1 alpha and IL-1 beta on tumorigenicity patterns and invasiveness

In this study, we show that distinct compartmentalization patterns of the IL-1 molecules (IL-1α and IL-1β), in the milieu of tumor cells that produce them, differentially affect the malignant process. Active forms of IL-1, namely precursor IL-1α (pIL-1α), mature IL-1β (mIL-1β), and mIL-1β fused to a signal sequence (ssIL-1β), were transfected into an established fibrosarcoma cell line, and tumorigenicity and antitumor immunity were assessed. Cell lines transfected with pIL-1α, which expresses IL-1α on the membrane, fail to develop local tumors and activate antitumor effector mechanisms, such as CTLs, NK cells, and high levels of IFN-γ production. Cells transfected with secretable IL-1β (mIL-1β and ssIL-1β) were more aggressive than wild-type and mock-transfected tumor cells; ssIL-1β transfectants even exhibited metastatic tumors in the lungs of mice after i.v. inoculation (experimental metastasis). In IL-1β tumors, increased vascularity patterns were observed. No detectable antitumor effector mechanisms were observed in spleens of mice injected with IL-1β transfectants, mock-transfected or wild-type fibrosarcoma cells. Moreover, in spleens of mice injected with IL-1β transfectants, suppression of polyclonal mitogenic responses (proliferation, IFN-γ and IL-2 production) to Con A was observed, suggesting the development of general anergy. Histologically, infiltrating mononuclear cells penetrating the tumor were seen at pIL-1α tumor sites, whereas in mIL-1β and ssIL-1β tumor sites such infiltrating cells do not penetrate inside the tumor. This is, to our knowledge, the first report on differential, nonredundant, in vivo effects of IL-1α and IL-1β in malignant processes; IL-1α reduces tumorigenicity by inducing antitumor immunity, whereas IL-1β promotes invasiveness, including tumor angiogenesis, and also induces immune suppression in the host.

Thymus-Derived Lymphocytes: Humoral and Cellular Reactions Distinguished by Hydrocortisone

Lymphocytes derived from the thymus (T cells) take part in the induction of humoral antibody and also effect cell-mediated graft-versus-host reactions. Preliminary treatment of mice with hydrocortisone caused an inhibition of T-cell function in humoral immunity, while enhancing the graft-versus-host reactivity of the same population of spleen cells. This suggests that different types of T cells participate in cellular and humoral immune reactions.

Nonmetastatic tumor cells acquire metastatic properties following somatic hybridization with normal cells.

SummarySomatic cell hybridization between nonmetastatic tumor cells and normal cells of the lymphoreticular system results in hybrid cells manifesting metastatic properties of defined target organ specificity. Thus, fusion of the nonmetastatic BALB/c originated NSI plasmacytoma with C57BL B lymphocytes resulted in hybridomas, each of which were metastatic. Of 10 hybridomas, 7 generated metastases in the spleen and liver, whereas 3 generated liver metastases. The generation of liver metastases by hybridomas which homed to both spleen and liver, but not by those which homed to the liver only, was controlled by the spleen. The acquisition of metastatic properties via somatic cell fusion seems to represent a general principle, in which the normal partner determines the target organ specificity for the metastatic growth. Thus, fusion of SP2/O myeloma cells with syngeneic B lymphocytes also resulted in a hybrid cell metastasizing to the spleen and liver, yet a somatic hybrid between NSI and a macrophage or dendritic-like cell metastasized to the lung. Cell surface molecules encoded by the genome of the normal partner was demonstrated to control the target organ specificity: antibodies against MHC-encoded antigens of the normal B cell partner prevented the generation of metastases by hybridomas metastasizing to the spleen and liver, but not by those metastasizing to the liver only. This is in accordance with the function of MHC molecules on lymphocytes in controlling their homing to lymphoid organs. Hybridomas of T cell lymphomas also manifested metastatic properties. Analysis of the cell surface Thy-1 antigens of a hybridoma (DCH10), produced via somatic fusion between BW5145 lymphoma and a putative macrophage cell indicated that cells of liver metastases (DCH10-Li) generated by the hybrid cells might have undergone further somatic cell fusion in vivo with host (T?) cells. These cells have acquired new metastatic properties, generating metastases in spleen, liver and kidneys. In fact, even the inoculation of the parental BW lymphoma cells resulted in a case of liver metastasis (BW-Li). Such BW-Li cells, upon reinoculation, also generated metastases in the spleen, liver and kidneys. Analysis of the Thyl phenotype indicated that BW-Li cells may also have undergone somatic cell fusion in vivo with host (T?) cells, resulting in the acquisition of metastatic properties. The pattern of cell-cell interactions (adhesion, infiltration) with liver cell monolayers of BW-Li cells and of DCH10-Li (T-cell lymphomas) was identical, and differed from cells of liver metastases of the myeloma-B cell hybridomas which might be based on responses to liver growth signals. Accordingly, the morphology of liver metastases generated by the two categories of hybridomas was different. It appears therefore, that (a) the acquisition of metastatic properties following somatic cell fusion with normal lymphoreticular cells is of a general significance; (b) somatic cell fusion provides an experimental system for the analysis of molecular properties determining the acquisition of metastatic capability; and (c) it may also represent a mechanism for tumor progression in vivo.

Selection of 3LL tumor subline resistant to natural effector cells concomitantly selected for increased metastatic potency

SummaryStudies were carried out to test whether the selection of 3LL tumor cells resistant to the cytotoxic activity of normal spleen cells will concomitantly select for increased metastatic capacity. A total of 0.5×106 3LL tumor cells and 50×106 normal spleen cells were admixed and inoculated SC in C57BL/6 mice. This procedure was repeated for eight transplant generations. The tumor cells which were selected in this manner (3LLN) were relatively resistant in vitro to the cytotoxic effect of normal spleen sells. The 3LLN cells were less susceptible than the original 3LL tumor cells to the hybrid resistance mechanisms, as determined by their growth capacity in F1(BALB/c×C57BL/6) or F1(C3H/DiSn×C57BL/6) mice. After three selection generations, 3LLN tumor cells were more efficient in the production of spontaneous lung metastases. These results support our suggestion that natural cell-mediated immunity may be involved in the control of metastatic spread.

Acute infection of mice with lactic dehydrogenase virus (LDV) impairs the antigen-presenting capacity of their macrophages

Abstract The infection of mice with lactic dehydrogenase virus (LDV) is characterized by elevated levels of various plasma enzymes such as lactic dehydrogenase, malic dehydrogenase, and others. This elevation is probably the consequence of a defect in the clearance capacity of the virus-affected reticuloendothelial cells, which were found to serve as the targets for LDV infection. Since macrophages play a pivotal role in the induction and regulation of cellular immune responses, we tested the antigen-presenting capacity of macrophages from LDV-infected mice, using a system in which in vitro reactivation of memory T cells depends on specific antigen presentation by macrophages. Our experiments revealed that the antigen-presenting capacity of spleen, lymph node, and peritoneal antigen-presenting macrophages from LDV-infected mice was impaired. This impairment, however, was not due to a defective cellular concentration capacity of antigen, since no difference in the uptake of radiolabeled antigen by uninfected and acutely LDV-infected macrophages was observed. Similarly one cannot attribute the impaired presentation capacity to suppressor cells, since we found that LDV-infected macrophages are not differentially immunosuppressive in the specific in vitro assays used. The analysis of peritoneal macrophages for their expression of Ia antigens revealed that the proportion of Ia-positive macrophages among the LDV-infected peritoneal cells is reduced in comparison to their proportion in noninfected mice. Our results suggest, therefore, that infection of macrophages by LDV is followed by an impairment of their antigen-presenting capacity, probably due to a reduction in the relative proportion of Ia-positive macrophages. These results indicate that the virus either impairs the expression of membrane-associated antigen-presenting components (such as the Ia determinants), thus damaging antigen presentation, or is responsible for the elimination of Ia-positive cells from the peritoneum.

MANIPULATION OF METASTASIS AND TUMOUR GROWTH BY TRANSFECTION WITH HISTOCOMPATIBILITY CLASS I GENES

Approximately 50% of fibrosarcomas induced with methylcholanthrene A were found to be defective in H‐2 expression. In tumours which lack H‐2K antigens, H‐2 gene transfection was used to restore H‐2K expression. The de novo expression of H‐2K reduced tumorigenicity and abolished the formation of metastases in syngeneic mice. Expression of H‐2K seems to render the tumour cells immunogenic and leads to effective recognition of the tumour cells by the host immune system.