Alcohol consumption decreases IL-2-induced NF-kappaB activity in enriched NK cells from C57BL/6 mice.

Previously we showed that ethanol (EtOH) consumption suppressed IL-2-induced cytolytic activity of murine splenic natural killer (NK) cells. Although IL-2 receptor signaling is involved in activation of NK cells, neither the mechanism for this activation nor the role of EtOH consumption in modulating activation is completely understood. In this study we show by electrophoretic mobility-shift assay (EMSA) that enriched splenic NK cells from EtOH-consuming C57BL/6 mice exhibit reduced NF-kappaB and AP-1 binding activity in response to IL-2 stimulation as compared to the water-drinking mice. Semiquantitative RT-PCR and real-time PCR analyses indicated that EtOH consumption inhibits the induction of perforin, granzyme A, and granzyme B in response to IL-2. Pyrrolidine dithiocarbamate (PDTC) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) blocked NFkappaB and AP-1 binding activity in nuclear extracts of IL-2-stimulated NK cells in an EMSA and also inhibited the IL-2-induced expression of perforin, granzyme A, and granzyme B gene expression in enriched NK cells. These inhibitors dramatically suppressed IL-2-stimulated NK cytolytic activity against YAC-1 lymphoma target cells. Taken together, these results suggest that NFkappaB and AP-1 are important regulators of NK cell cytolytic function through regulation of perforin, granzyme A, and granzyme B gene expression. The findings further suggest that the decreased cytolytic activity of IL-2-stimulated NK cytolytic activity in EtOH-consuming mice is due at least in part to impaired transactivation of these and possibly other genes involved in control of NK-cell target lysis.

Specific amino acid deficiency alters the expression of genes in human melanoma and other tumor cell lines.

This study determined the effect of tyrosine (Tyr) and phenylalanine (Phe) deprivation on protein expression and phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4)/stress-activated protein/Erk kinase (SEK1), a metastasis suppressor gene. Differential display and suppressive subtractive hybridization techniques identified genes modulated by Tyr and Phe deprivation. Expression of MKK4/SEK1 protein varied widely among human A375, A375SM and SB2 melanoma, PC-3 and DU145 prostate cancer, and MDA-MB-231 breast cancer cell lines and within the different lines. Phosphorylation of the MKK4/SEK1 protein similarly varied. No differences in MKK4/SEK1 gene expression or in the 41 other metastasis and tumor suppressor genes were found in A375 melanoma cells cultured in Tyr- and Phe-deprived media. A number of up-regulated and down-regulated genes in A375 melanoma cells were identified by differential display and suppressive subtractive hybridization that were pertinent to regulation of cytoskeletal organization, cell movement, gene transcription and metastasis. Two tumor marker genes, the gene for enolase and FUS/CHOP, were down-regulated by Tyr and Phe deprivation. This study shows that tumor cells display heterogeneity in their response to deprivation of Tyr and Phe and that these amino acids may be signaling molecules that regulate gene expression and function in tumor cells.

Chronic ethanol inhibits NK cell cytolytic activity: role of opioid peptide beta-endorphin.

The role of beta-endorphin (beta-EP) in ethanol-altered NK cell cytolytic activity is studied using male Fischer-344 rats as an animal model. Ethanol was administered for 1, 2, 3, or 4 wk in a liquid diet containing 8.7% ethanol (v/v), which means that 37% of the total calories were derived from ethanol. Rats treated with ethanol for 1 wk showed an increase in hypothalamic and plasma levels of immunoreactive (IR)-beta-EP, but displayed no significant effect on NK cell activity determined by (51)Cr release assay, as compared with those in pair-fed and ad libitum-fed animals. However, animals treated with ethanol for 2, 3, or 4 wk showed decreased hypothalamic and plasma levels of IR-beta-EP and decreased splenic NK cell activity. No significant decrease in the number of splenocytes and NK cells or in the percentage of NK cells was seen until after 3 and 4 wk of ethanol treatment. Exposure in vitro of splenic lymphocytes obtained from control animals to various concentrations of beta-EP increased NK cell activity. The opiate antagonist naltrexone blocked the beta-EP-stimulated effect. The in vitro NK cell response to beta-EP was reduced in the splenocytes obtained from animals treated with ethanol for 2 wk, but not in those obtained from animals treated with ethanol for 1 wk as compared with those in control animals. Additionally, beta-EP administration into the paraventricular nucleus of the hypothalamus stimulated NK cell cytolytic activity, whereas the opiate blocker administration reduced NK cell activity. The NK cell responses to paraventricular nucleus beta-EP were reduced in the animals treated with ethanol for 2 wk. These data provide evidence for the first time that ethanol inhibits NK cell cytolytic activity, possibly by reducing beta-EP-regulated splenic NK cell function.

UCN-01 dose-dependent inhibition of normal hyperproliferative cells in mice.

UCN-01 is a hydroxylated derivative of staurosporine and a potent protein kinase C (PKC) inhibitor. Interest in the potential usefulness of this compound as an anticancer drug stems mainly from its unique anti-signaling, growth-arresting properties on tumor cells. This include activation of CDC2 kinase (CDK1) which interacts with either cyclin A or cyclin B1 at the G1 or G2/M border, suggeting that this event is one of the major consequences of the drug action on eukaryotic cells. Nonetheless, the antiproliferative activity of UCN-01 on normal rapidly dividing cells (intestinal epithelial and bone marrow cells) is not well documented. Thus, the main objective of this study was to investigate the in vivo antiproliferative activity of UCN-01 on these normal hyperproliferative cells and evaluate whether cellular response to UCN-01 could be modulated in the presence of DNA damage. Mice were injected i.m. with a single dose of UCN-01 (2.5 mg/kg-20 mg/kg) followed 3 and 24 h later by in vivo BrdU labeling for 1 h. At autopsy, bone marrow cells were collected and fixed for dual parameter BrdU/DNA flow cytometry. Different regions of the gut were also fixed for immunoperoxidase BrdU assays. Newly replicated cells were mainly located in the lower compartments of the crypt columns and were scored for BrdU stained nuclei using an image analysis system. A comparison between groups showed that 5 mg/kg UCN-01 induced inhibition in BrdU incorporation at 3 and 24 h, as compared to the other groups injected with various doses of UCN-01. Flow cytometric analysis of bone marrow cells stained with fluorescein tagged anti-BrdU (FITC) along with propidium iodide (PI) also showed inhibition in BrdU incorporation of S phase fraction cells in mice treated with 5 mg/kg UCN-01. These bone marrow cells were arrested primarily in the G1 phase of the cell cycle. The colony-forming unit (CFU) assay of the bone marrow cells was then used to determine the level of drug interaction of UCN-01 and, topotecan, a topoisomerase I inhibitor, at a fixed dose ratio. An antagonistic drug interaction (CI > 1) was observed as determined by the median-effect analysis. However, an additive interaction (CI = 1) was obtained with the use of camptothecin or 10,11-methylenedioxycamptothecin and UCN-01. The results of the in vitro drug interaction with UCN-01 may predict protection from topotecan-induced bone marrow toxicity.

Tob-mediated cross-talk between MARCKS phosphorylation and ErbB-2 activation.

The biochemical path for the activation of ErbB-2 by PKC activator was investigated in MDA-MB-231 human breast cancer cells. We found that PMA-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) increased its binding with Tob that exerts an anti-proliferative effect through the binding with ErbB-2. The phosphorylation site domain (PSD) of MARCKS was relevant to its interaction with Tob. Decreased binding of Tob with ErbB-2 and subsequent activation of ErbB-2 were observed in MDA-MB-231 cells in response to PMA treatment. The present study proposes that MARCKS phosphorylation by PKC removes Tob from ErbB-2 by increasing its binding affinity with Tob, and thereby activates the ErbB-2 mediated signal transduction.

Decreased tissue plasminogen activator and increased plasminogen activator inhibitors and increased activator protein-1 and specific promoter 1 are associated with inhibition of invasion in human A375 melanoma deprived of tyrosine and phenylalanine.

We previously found that dietary tyrosine (Tyr) and phenylalanine (Phe) restriction significantly decreased the metastatic phenotype of the pigmented murine B16BL6 melanoma in vivo and decreased the in vitro invasion of these cells. Here we report that invasion and chemoinvasion through GFR Matrigel of the human amelanotic A375 melanoma also is significantly inhibited by Tyr and Phe deprivation in vitro. Deprivation of these two amino acids decreased the secretion and protein expression of tissue-type plasminogen activator (tPA) while expression and secretion of plasminogen activator inhibitor (PAI-1 and PAI-2) were increased. Moreover, nuclear extracts of Tyr- and Phe-deprived cells exhibited increased binding of the transcription factors, activator protein-1 (AP-1) and specific promoter-1 (Sp1), to consensus oligonucleotides as determined by electrophoretic mobility shift assay. Nuclear binding activity to the oligonucleotide consensus sequence for AP-1 was inhibited by antibody against c-Fos and more effectively inhibited by an antibody against c-Jun. We conclude that decreased invasion and chemoinvasion of A375 melanoma cells deprived of Tyr and Phe are related to decreased secretion of tPA and increased secretion of PAIs. Increased AP-1 and Sp1 binding implicates these transcription factors in the regulation of PAI expression.

The modulation of B16BL6 melanoma metastasis is not directly mediated by cytolytic activity of natural killer cells in alcohol-consuming mice.

BACKGROUND: Previous studies in our laboratory indicate that alcohol consumption suppresses the metastasis of B16BL6 melanoma, whereas the cytolytic activity of natural killer (NK) cells is decreased in female C57BL/6 mice given 20% w/v alcohol in their drinking water. In the present study, we further evaluated the involvement of NK cells and alcohol consumption in the cytolytic activity of NK cells, the surface expression of NK phenotypic markers, and metastasis of B16BL6 melanoma in C57BL/6 beige (bgJ/bgJ) mutant mice, which possess inherently low NK-cell cytolytic activity. METHODS: Beige and control (bgJ/+) mice were given either water or 20% w/v of alcohol in drinking water for 6 1/2 to 7 weeks before assay for cytolytic activity, surface marker expression, and inoculation with B16BL6 melanoma intravenously or into the pinna of the ear. RESULTS: NK cytolytic activity was suppressed in beige mice, and alcohol consumption did not modulate further the cytolytic activity. Beige mice had a lower percentage of NK cells in the peripheral blood and spleen than control mice. Peripheral blood lymphocytes from beige mice also exhibited a reduced percentage of CD4+ T lymphocytes. Alcohol consumption similarly reduced the percentages of NK1.1- and LGL-1-expressing lymphocytes in the peripheral blood and spleen and reduced the percentage of CD8+ T lymphocytes in the peripheral blood in both control and beige mice. Tumor lung colonization was increased in beige mice relative to control mice after intravenous inoculation of B16BL6 melanoma. The increase was more pronounced in water-drinking beige mice than in control mice irrespective of alcohol consumption. Tumor lung colonization was significantly decreased (p < 0.05) by alcohol consumption in one experiment and partially decreased (p = 0.07) in the other. Mice that were inoculated into the pinna of the ear also exhibited a blunted antimetastatic response to alcohol consumption. CONCLUSIONS: These data suggest that the presence of the beige mutation diminishes the antimetastatic effect of alcohol consumption and that there is no interaction between alcohol consumption and NK-cell activity in the modulation of lung metastasis of B16BL6 melanoma cells.

Focal adhesion kinase-dependent apoptosis of melanoma induced by tyrosine and phenylalanine deficiency.

We found previously that restriction of tyrosine (Tyr) and phenylalanine (Phe) inhibited growth and metastasis of B16BL6 murine melanoma and arrested these cells in the G0-G1 phase of the cell cycle. Here, we report that deprivation of these two amino acids in vitro induces apoptosis in B16BL6 and in human A375 melanoma cells but not in nontransformed, neonatal murine epidermal cells or human infant foreskin fibroblasts. Four days after deprivation of Tyr and Phe in vitro, 37% of B16BL6 and 51% of A375 melanoma cells were undergoing apoptosis. Apoptosis was not associated with elevation in intracellular calcium or alteration in p53 or c-myc protein expression. Expression and Tyr phosphorylation of focal adhesion kinase (FAK) were inhibited in both melanoma cell lines by deprivation of Tyr and Phe but not by deprivation of glutamine or serum. Tyr phosphorylation of FAK in Tyr- and Phe-deprived melanoma cells was enhanced within 30 min of refeeding with complete DMEM. FAK protein expression recovered within 60 min, and cell viability recovered within 24 h. Genistein, a tyrosine kinase inhibitor that specifically inhibits Tyr phosphorylation of FAK, did not induce apoptosis in A375 melanoma cells at a concentration of 50 microM. Genistein prevented the recovery of cell viability upon refeeding with Tyr and Phe to previously deprived A375 melanoma cells. These data collectively indicate that apoptosis induced by Tyr and Phe deprivation is FAK-dependent.

Inhibition of B16BL6 melanoma invasion by tyrosine and phenylalanine deprivation is associated with decreased secretion of plasminogen activators and increased plasminogen activator inhibitors.

We previously found that dietary tyrosine (Tyr) and phenylalanine (Phe) limitation significantly decreased the metastatic phenotype of B16BL6 melanoma cells in vivo and decreased the in vitro invasion of these cells. To more specifically characterize the effects of Tyr and Phe deprivation we examined the three steps involved in invasion: attachment to host cells and components, elaboration of proteases that degrade basement membranes, and migration of invading tumor cells. Here we report that B16BL6 melanoma cell invasion through growth factor reduced (GFR) Matrigel is significantly decreased by Tyr and Phe deprivation. Tyr and Phe deprivation in vitro decreased the attachment of B16BL6 melanoma cells to GFR Matrigel, heparin sulfate proteoglycans (HSPG), neonatal murine epidermal (NME) cells and the extracellular matrix (ECM) from these cells. These cells also exhibited a decrease in chemotactic response to fetal bovine serum (FBS). Deprivation of these two amino acids decreased the secretion of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) while plasminogen activator inhibitor (PAI)-1 and -2 were increased in these cells. These observations suggest that Tyr and Phe deprivation decreases the in vitro chemotactic and invasive ability of B16BL6 melanoma cells by decreasing attachment and secreted PA activity and by increasing secreted PAIs in these cells.

Influence of tyrosine and phenylalanine limitation of cytotoxicity of chimeric TGF-alpha toxins on B16BL6 murine melanoma in vitro.

Previous research in animals supports the use of tyrosine and phenylalanine (Tyr-Phe) restriction as an adjuvant to the treatment of cancer. In this regard, dietary restriction of Tyr-Phe specifically inhibits the growth of B16BL6 melanoma tumors, dramatically suppresses spontaneous hematogenous metastasis, and modulates the sensitivity of these tumor cells to growth factors. Two chimeric toxins, HB-TGF alpha-PE4EKDEL and TGF alpha-PE4EKDEL, were examined for their toxicity against the B16BL6 melanoma cell line, and the ability of Tyr-Phe limitation to modulate the potential of these toxins was examined. Tyr-Phe limitation significantly enhanced the cytotoxic effects of HB-TGF alpha-PE4EKDEL approximately 10-fold toward B16BL6 melanoma, and free heparin diminished the cytotoxicity of HB-TGF alpha-PE4EKDEL. Although TGF alpha-PE4EKDEL is cytotoxic to this cell line, Tyr-Phe limitation did not effect the cytotoxicity of this toxin. Tyr-Phe limitation inhibited the synthesis and secretion of heparin-binding proteins but did not alter the expression of surface heparan sulfate proteoglycans. These data suggest that cell surface heparan sulfate proteoglycan is a target for binding and execution of the cytotoxicity of HB-TGF alpha-PE4EKDEL and that augmentation of cytotoxicity by Tyr-Phe limitation is due to the inhibition of heparin-binding protein production.

Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice.

This study was designed to investigate the effects of moderate-intensity endurance training on basal natural killer (NK) cell cytolytic activity in murine splenocytes that were enriched for 1) NK1.1+ cells or 2) macrophages and NK1.1+ cells. Mice were assigned to sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn) groups. Splenocyte number, the percentages of NK1.1+, large granular lymphocytes (NK1.1+, LGL-1+), and other subpopulations did not change in Trn mice. Approximately 70% of cells enriched for NK1.1+ expressed this surface antigen. Lytic units (LU) expressed per LGL-1+ cell were significantly lower in Trn [83.9 +/- 3.2 (SE)] compared with Sed (109.5 +/- 7.5) and TM (101.3 +/- 6.4) groups. When macrophages remained in the in vitro assay, LU per LGL-1(+) cell did not differ across groups. The results indicate that highly enriched NK1.1+ cells from Trn mice had lower NK cell activity compared with Sed mice. No differences in NK cell activity were observed when cells were enriched for NK1.1+ cells and macrophages. These findings support the hypothesis that macrophage modulation of NK cells may be one mechanism contributing to augmented basal NK cell activity in endurance-trained individuals.

Tyrosine and phenylalanine restriction induces G0/G1 cell cycle arrest in murine melanoma in vitro and in vivo.

Tyr-Phe and Met limitation in vitro inhibited cell proliferation and proliferating cell nuclear antigen (PCNA) expression to a greater extent than serum limitation. Tyr-Phe and serum limitation arrested cells in the G0/G1 phase; Met limitation blocked cells in the G0/G1 and S phases. Tyr-Phe limitation progressively decreased cyclin D1 expression to 30% of control within four days and did not affect expression of cyclin D3 or cyclin-dependent kinase (CDK2, CDK4, and CDK5) expression, Met limitation decreased cyclin D3 expression to 25% of control and CDK2 expression to 32% of control by Day 4 and did not affect expression of cyclin D1, CDK4, and CDK5. Serum limitation inhibited cyclin D1 and cyclin D3 expression to 24% of control after four days and did not effect CDK expression. Expression of two CDK inhibitors, p21WAF1/Cip1 and p27Kip1, was not changed by amino acid or serum limitation. Dietary restriction of Tyr-Phe in mice bearing subcutaneous B16BL6 melanoma tumors decreased tumor growth rate compared with mice fed a normal diet. Tumors from Tyr-Phe-restricted mice exhibited decreased PCNA expression, G0/G1 phase cell cycle arrest, and reduced cyclin D1 expression. These data indicate that decreased tumor growth in vivo associated with dietary restriction of Tyr and Phe is cell cycle specific.

Ethanol modulates metastatic potential of B16BL6 melanoma and host responses.

The experimental metastatic potential (lung-colonizing ability) of B16BL6 melanoma cells was examined in C57BL/6 mice after exposure to ethanol in vitro and in vivo. In vitro, tumor cells were cultured with ethanol (0.3% v/v), or medium alone, for three passages at 5-day intervals. In vivo, B16BL6 melanoma was exposed to ethanol by administering ethanol (10% or 20% w/v) to mice following subcutaneous inoculation of tumor cells into the dorsal hip. All tumor cells were subsequently inoculated intravenously into the lateral tail vein of water-drinking mice to assess changes in metastatic phenotype. Tumor cells cocultured in vivo with ethanol produced significantly higher numbers of superficial lung colonies, compared with tumor cells cultured in control medium. Experimental metastasis of tumor cells obtained from 20% w/v ethanol-consuming mice was also significantly increased, compared with cells obtained from water-drinking mice. Metastasis of B16BL6 melanoma cells previously obtained from mice consuming 10% w/v ethanol did not differ from controls. In other experiments, water-drinking and ethanol-consuming (2.5%, 10%, and 20% w/v) mice were inoculated subcutaneously into the dorsal hip with B16BL6 melanoma cells, and monitored for tumor growth rate and survival time. In these experiments, survival times were significantly shorter in mice consuming 20% ethanol, compared with all other groups. Subcutaneous tumor growth rate was unaffected by ethanol consumption. Lung metastasis resulting from subcutaneous tumor implantation of B16BL6 melanoma was respectively inhibited, or absent, in 10% and 20% ethanol-consuming groups. Thus, tumor growth rate and incidence of lung metastases were not apparent determinants of decreased survival in 20% ethanol-consuming mice. The results of this study indicate that the experimental metastatic potential of B16BL6 melanoma is increased during exposure to ethanol; however, metastasis from subcutaneous tumor-bearing mice is suppressed. This latter finding is consistent with previous results in which spontaneous metastasis was also suppressed after inoculation of the tumor into the pinna of the ear. Although ethanol increases the ability of B16BL6 melanoma to colonize the lung after intravenous inoculation, this effect is abated in the presence of host factors in ethanol-consuming mice.

Ethanol consumption suppresses the IL2-induced proliferation of NK cells.

Ethanol (20% w/v) given to female, C57BL/6 mice in their drinking water suppresses natural killer (NK) and lymphokine activated killer cell cytolytic activity in mixed splenocytes and in splenocytes highly enriched for NK cells. The present study examined the effects of ethanol consumption on rIL2-induced proliferation of enriched NK cells. Mice were given 20% w/v ethanol in the drinking water for 2 weeks. Splenic NK cells were harvested and enriched up to 88% based on surface expression of NK1.1. The enriched NK cells were cultured in the presence of 1000 IU/ml (20 pg/ml) murine recombinant interleukin 2 (rIL2). There were fewer cells (p < 0.02) from ethanol-consuming mice compared to cells from water-drinking control mice after incubation with IL2 at 2, 4, and 6 days of culture. Ethanol consumption was associated with significantly lower [3H]thymidine uptake (p < 0.05). Ethanol consumption did not affect apoptosis or intracellular levels of interferon-gamma, tumor necrosis factor-alpha, or granulocyte macrophage colony-stimulating factor in NK cells. Ethanol consumption did not affect the expression of c-myc mRNA in NK cells that were cultured for 10 min or 4, 8, and 18 hr in rIL2. Suppression of IL2-induced NK cell proliferation is associated with ethanol consumption, and suppression is not due to altered IL2 receptor expression, increased apoptosis, intracellular cytokine levels, or c-myc expression.

Phorbol 12-myristate 13-acetate enhances nm23 gene expression in murine melanocytes but not in syngeneic B16-BL6 melanoma variants.

The nm23 gene has been described as a potential metastasis suppressor gene in certain rodent and human tumors. We previously demonstrated that tyrosine and phenylalanine restriction suppresses metastatic heterogeneity of B16-BL6 murine melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigated nm23 expression in the highly metastatic B16-BL6 (ND) melanoma, its nutritionally derived poorly metastatic (LT) variant, and the syngeneic non-tumorigenic Mel-ab melanocytes. No differences in nm23 expression were observed between ND and LT cells, and nm23 expression varied between different isolates. Previously, we showed that metastatic potential of 1-ND cells decreases and is not altered in 1-LT cells after prolonged in vitro cell passage; however, nm23 expression is equivalently increased by 2-fold. In 2-ND and 2-LT cells, expression of nm23 is not different at higher in vitro cell passage. Expression of nm23 decreased about 2-fold when phorbol 12-myristate 13-acetate (PMA) was removed from Mel-ab cells, which induces these cells to become quiescent. Although membrane-associated protein kinase C (PKC) activity decreased after prolonged PMA treatment in all cells, neither nm23 expression nor proliferation of ND and LT cells was affected by PMA. These data indicate that nm23 expression is related to proliferative activity rather than to the suppression of metastatic potential.

Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine.

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

Tyrosine and phenylalanine restriction sensitizes adriamycin-resistant P388 leukemia cells to adriamycin.

Cancer chemotherapy frequently fails, because tumors develop multiple drug resistance (MDR). Pharmacological efforts to reverse this MDR phenotype and sensitize resistant tumor cells have utilized verapamil (VER) to inhibit p-glycoprotein function and buthionine sulfoximine (BSO) to inhibit glutathione synthesis. Our previous results indicate that restriction of two amino acids, tyrosine (Tyr) and phenylalanine (Phe), may potentially suppress the MDR phenotype. These results show that in vivo Tyr and Phe restriction improves the therapeutic response of a metastatic variant of B16-BL6 (BL6) murine melanoma to adriamycin (ADR) and B16 melanoma to levodopa methyl ester. We examine whether in vitro limitation of Tyr and Phe suppresses ADR resistance of BL6 cells and whether Tyr-Phe modulation of the MDR phenotype is applicable to other tumor types, particularly P388 murine leukemia. Mechanisms underlying Tyr-Phe modulation of ADR resistance are examined in the presence of VER and BSO, singly and in combination. Our results indicate that in vitro Tyr and Phe restriction has no effect on BL6 resistance to ADR. However, Tyr and Phe restriction does increase the sensitivity of ADR-resistant P388 cells to ADR without affecting drug efflux, ADR uptake, or glutathione levels. In addition, this enhanced ADR sensitivity of P388 cells is even more pronounced in the presence of BSO. Suppression of ADR resistance in P388-resistant cells by Tyr and Phe restriction indicates that Tyr- and Phe-mediated modulation of the MDR phenotype is possible and that Tyr and Phe restriction may be useful as a potential adjuvant to effective cancer chemotherapy.

Differences in expression of metalloproteinases and plasminogen activators in murine melanocytes and B16 melanoma variants: lack of association with in vitro invasion.

We investigated in vitro chemotactic responses to fibronectin and laminin, invasion through reconstituted basement membrane (Matrigel) and secretion of matrix metalloproteinases and plasminogen activators by non-tumorigenic Mel-ab melanocytes; B16 melanoma; and the metastatic sublines, B16F1, B16F10 and B16BL6. In vitro chemotactic and invasive ability were not associated with in vivo metastatic potential. Secretion of various matrix-degrading enzymes was not related to in vitro invasion. Conditioned media from all B16 melanoma sublines, but not from Mel-ab cells, contained the M(r) 92,000 progelatinase. The activated M(r) 85,000 species was present only in conditioned media from Mel-ab, B16 and B16F1 cells. Mel-ab cells secreted copious amounts of the M(r) 72,000 progelatinase, and the M(r) 66,000 active form was also present in conditioned media. Secretion of the M(r) 72,000 progelatinase by B16 melanoma sublines was markedly lower, and only conditioned media from B16 cells contained the activated M(r) 66,000 form. Furthermore, cell lysates of Mel-ab cells contained a M(r) 67,000 metalloproteinase which was absent in the tumor cells. All cells secreted tissue plasminogen activator; however, the metastatic B16F1, B16F10 and B16-BL6 cells also secreted urokinase plasminogen activator. Our results indicate that matrix metalloproteinase secretion by itself is not associated with tumorigenicity or metastatic potential. Secretion of urokinase plasminogen activator, and not tissue plasminogen activator, reflected the metastatic characteristics of the B16 melanoma tumor sublines.

Ethanol consumption reduces the cytolytic activity of lymphokine-activated killer cells.

Ethanol (20% w/v) given to female C57BL/6 mice in their drinking water reduces splenic natural killer (NK) cell cytolytic activity after 2, 4, and 10 weeks of consumption. This reduction is transient because the levels of NK cell cytotoxicity from ethanol-consuming mice are nearly equal to those of water-drinking mice after splenocytes were incubated in 1000 IU/ml of recombinant interleukin-2 (rIL2) for 16-18 hr. In this study, mice were given 20% w/v ethanol in the drinking water for 2 weeks. Splenic NK cells were enriched up to 88% by negative selection based on surface expression of NK1.1. Enriched NK cells were expanded in rIL2 for 6 days. Lymphokine-activated killer (LAK) cells from both ethanol-consuming and water-drinking mice were > 95% NK1.1+. LAK cell cytolytic activity was significantly lower against NK-insensitive P815 mastocytoma [6.67 +/- 2.18 vs. 17.21 +/- 1.8 lytic units (LUs), p < 0.01], moderately NK-sensitive B16 melanoma (25.3 +/- 6.6 vs. 66.2 +/- 14.2 LU, p < 0.05), and NK-sensitive YAC-1 lymphoma targets (80.5 +/- 34.7 vs. 177.0 +/- 43.6 LU, p < 0.005) in cells from ethanol-consuming mice compared with water-drinking controls. Ethanol consumption did not affect the morphology or phenotype of LAK cells with respect to surface expression of NK1.1, B220, CD3, CD25, CD11a, CD54, CD45RB, or class I major histocompatibility complex.