The efficacy of 2',2'-difluorodeoxycytidine (gemcitabine) and vinblastine combined with interferon in nude mice xenografts of human renal cell carcinoma

Recent in vitro experiments indicated strong activity of 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) in human renal cell carcinoma (RCC) cell lines and an increase of efficacy by combined application of interferon (IFN). In the present study, nude mice with xenografts from ACHN- or SN12C cells were treated by dFdC, dFdC plus IFN-alpha or vinblastine (VBL) plus IFN-alpha. ACHN-xenografts were significantly more inhibited by dFdC+/-IFN-alpha than by VBL+IFN-alpha. Complete remissions (CR) were only seen by dFdC. An additional treatment with IFN-alpha shortened the time to commencement of tumor remission and increased CR of ACHN- and SN12C-tumors (40%; 7%) compared to a treatment with dFdC alone (20%; 0). dFdC+IFN-alpha reduced the number of pulmonary metastases compared to untreated animals. Survival was significantly prolonged by dFdC+/-IFN-alpha in ACHN-mice and dFdC+IFN-alpha or VBL+IFN-alpha in SN12C mice. In conclusion, experimental data confirm dFdC as a superior drug against human RCC compared to VBL. Combined therapy with IFN-alpha increased the efficacy of dFdC in terms of tumor response in immunodeficient nude mice, thus clinical studies are strongly recommended in patients with metastatic renal cell carcinoma.     

Extracorporeal shock wave lithotripsy for lower pole nephrolithiasis: efficacy and variables that influence treatment outcome

OBJECTIVES: To clarify the role of the membranous glycoprotein gp-170 in renal cell carcinoma (RCC) cell lines and their multidrug resistant (MDR) sublines. and to correlate gp-170 with the natural and acquired drug resistance of these cell lines to anthracyclines. MATERIALS AND METHODS: The expression of gp-170 in five cultured RCC cell lines and serial RCC8701 MDR sublines was analysed by immunofluorescent flow cytometry. The chemosensitivity of these tumour cells to the anthracycline anticancer drugs adriamycin and epirubicin was measured using the microplate tetrazolium (MTT) cytotoxicity assay, and the results correlated with gp-170 expression. RESULTS: All six natural RCC cell lines showed a variably increased expression of gp-170, with the A704 and Caki-1 cell lines the highest. In contrast, gp-170 expression increased and then was suppressed in acquired MDR sublines of RCC8701 cultured in increasing concentrations of adriamycin. The A704 and Caki-1 cells were much more resistant to adriamycin and epirubicin than the A498, ACHN and RCC8701 cell lines, in parallel with the expression of gp-170. The resistant cell line cultured long-term in 800 ng/mL adriamycin, RCC8701/ADR800, was 122 times more resistant to adriamycin and 238 times more resistant to epirubicin than the parent cell line: the pattern differed from that in native RCC cell lines and was unrelated to the expression of gp-170. CONCLUSION: Membranous gp-170 plays an important role in MDR of native RCC cell lines, while acquired MDR cells have different mechanisms of obtaining drug resistance in addition to gp-170. This phenomenon may be applicable to the clinical treatment of patients newly diagnosed with RCC or those with disease refractory to chemotherapy.     

p53 modulation of Fas/Apo-1 mediated apoptosis in a human renal cell carcinoma cell line

The mutant p53 gene was transfected into ACHN, a wild-type p53-containing human renal cell carcinoma (RCC) cell line. The colony forming efficiency in soft agar in the mutant-type p53-transfected cell line (ACHN/MP) was significantly higher than that in the vector-only transfected control cell line (ACHN/C). The anti-Fas monoclonal antibody (CH11) induced apoptosis in the ACHN/C cells in a dose-dependent manner, whereas the effect of CH11 on the ACHN/ MP cells was markedly suppressed. In addition, the cytotoxic effect of CH11 on the ACHN/MP cells was augmented by the pretreatment with interferon- , but the corresponding effect on ACHN/C cells was not. These findings suggest that Fas-mediated therapy could be a novel approach to RCC, if interferon- treatment is added according to the p53 gene status.     

Combination chemotherapy studies with gemcitabine and etoposide in non-small cell lung and ovarian cancer cell lines

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and etoposide (4'-demethylepipodo-phyllo-toxin-9-4,6-O-ethylidene-beta-D-g lucopyranoside, VP-16) are antineoplastic agents with clinical activity against various types of solid tumors. Because of the low toxicity profile of dFdC and the differences in mechanisms of cytotoxicity, combinations of both drugs were studied in vitro. For this purpose, we used the human ovarian cancer cell line A2780, its cis-diammine-dichloroplatinum-resistant and VP-16 cross-resistant variant ADDP, and two non-small cell lung cancer cell lines, Lewis Lung (LL, murine) and H322 (human). The interaction between the drugs was determined with the multiple drug effect analysis (fixed molar ratio) and with a variable drug ratio. In the LL cell line, the combination of dFdC and VP-16 at a constant molar ratio (dFdC:VP-16 = 1:4 or 1:0.125 after 4- or 24-hr exposure, respectively) was synergistic (combination index [CI], calculated at 50% growth inhibition = 0.7 and 0.8, respectively; CI <1 indicating synergism). After 24- and 72-hr exposure to both drugs at a constant ratio, additivity was found in the A2780, ADDP, and H322 cell lines (dFdC:VP-16 = 1:500 for both exposure times in these cell lines). When cells were exposed to a combination of dFdC and VP-16 for 24 or 72 hr, with VP-16 at its IC25 and dFdC in a concentration range, additivity was found in both the LL and H322 cells; synergism was observed in the A2780 and ADDP cells, which are the least sensitive to VP-16. Schedule dependency was found in the LL cell line; when cells were exposed to dFdC 4 hr prior to VP-16 (constant molar ratio, total exposure 24 hr), synergism was found (CI = 0.5), whereas additivity was found when cells were exposed to VP-16 prior to dFdC (CI = 1.6). The mechanism of interaction between the drugs was studied in more detail in the LL cell line; dFdCTP accumulation was 1.2-fold enhanced by co-incubation with VP-16, and was even more pronounced (1.4-fold) when cells were exposed to VP-16 prior to dFdC. dCTP levels were decreased by VP-16 alone as well as by the combination of both compounds, which may favor phosphorylation of dFdC, thereby increasing dFdCTP accumulation. DNA strand break (DSB) formation was increased for exposure to both compounds together compared to exposure to each compound separately, this effect being most pronounced when cells were exposed to VP-16 prior to dFdC (38% and 0% DSB for dFdC and VP-16 alone, respectively and 97% DSB for the combination). The potentiation in DSB formation might be a result of the inhibition of DNA repair by dFdC. Provided the right schedule is used, VP-16 is certainly a compound eligible for combination with dFdC.     

Characterization and mutual ligand-induced modulation of epidermal growth factor and interferon-alpha receptors on renal carcinoma cells in vitro

We have determined the binding of epidermal growth factor (EGF) and interferon (IFN)-alpha to their specific receptors on four renal carcinoma cell lines. CaKi-2, a-498 and ACHN cell lines express high numbers, and CaKi-1 expresses low number of EGF receptors (EGFRs). On all four renal carcinoma cell lines, we have also detected specific IFN-alpha binding sites. EGF and IFN-alpha binding to their receptors caused modulation of the other ligand's receptor binding. Scatchard analyses of binding data showed that IFN-alpha treatment leads to an increase of EGFR number in three out of four cell lines and to a decrease of EGFR number in one out of four (CaKi-1). No significant changes in EGF binding affinities were detected. EGF induced a reduction in IFN-alpha receptor number in all four cell lines without significant changes in IFN-alpha binding affinities. We hypothesize that presence of EGF in the microenvironment of renal cancer cells may modulate the biological effects of IFN and consequently decrease its antiproliferative activity.     

Cancer-associated expression of glycolipid sulfotransferase gene in human renal cell carcinoma cells

Human renal cell carcinoma (RCC) tissue and a cell line derived therefrom, SMKT-R3, showed markedly increased glycolipid sulfotransferase [cerebroside sulfotransferase (CST); EC 2.8.2.11] activity and accumulated sulfoglycolipids. Recently, we cloned a human CST cDNA from a SMKT-R3 cDNA library (K. Honke et al., J. Biol. Chem., 272: 4864-4868, 1997). In this study, we investigated the expression of the CST gene in seven human RCC lines (SMKT-R1, SMKT-R2, SMKT-R3, SMKT-R4, TOS-1, TOS-2, and ACHN) and their normal counterpart, human renal proximal tubular cells. On Northern blot analysis, a marked increase of CST mRNA was observed in every RCC line, except for ACHN, as compared with normal cells. ACHN cells showed a slightly increased level of CST mRNA. CST activity was correlated with the amount of mRNA. Sulfoglycolipid analysis revealed that expression of lactosylceramide sulfate was correlated with the CST level. Furthermore, we examined the effects of epidermal growth factor (EGF), tetradecanoylphorbol-13-acetate, and genistein, which are known to regulate CST activity in SMKT-R3 cells, on CST-gene expression in various RCC cells. On treatment with EGF, CST mRNA time-dependently increased in accord with its activity in SMKT-R3 cells. Yet, augmentation by EGF was only observed in SMKT-R3. In contrast, a reduction of CST mRNA and activity by tetradecanoylphorbol-13-acetate and genistein was observed in all of the lines examined. Taken together, these findings indicate that in human RCC cells, the CST gene is generally overexpressed via a signaling pathway involving protein kinase-C and tyrosine kinases.     

Modulation of associated ovarian carcinoma antigens by 5 cytokines used as single agents or in combination

Optimization of intraperitoneal radioimmunotherapy of ovarian cancer depends on increasing the antigenic expression of tumor cells. For this purpose, we studied the effect of 5 cytokines (IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha and TGF-beta), used as single agents or in combination, on 4 ovarian cancer cell lines which present different antigenic profiles with the monoclonal antibodies (MAbs) tested (OC125, OVTL-3, MOv 18 and MOv 19). Analyses were performed by flow cytometry and the Scatchard technique in order to study antigenic modulation. The effect on proliferation was determined by cell counting. Expression of O3 antigen, recognized by the OVTL3 MAb, was increased up to 2.5 times after IFNs and TNF-alpha (used as single agent) on the 2 lines presenting low basal expression (SHIN-3 and IGROVI). The expression of CA125 antigen and the antigens recognized by MOv 18 and MOv 19 MAbs was not increased by any of the cytokines tested. The combination IFN-gamma+TNF-alpha was synergistic on cytotoxicity and enhanced O3 expression, providing 10 times as many sites per cell on the SHIN-3 line. For 3 other associations (IFN-alpha+IFN-gamma, IFN-beta+IFN-gamma and IFN-alpha+TNF-alpha), there was an additive effect on O3 expression and on cell cytotoxicity.     

Lack of effect of different cytokines on expression of membrane-bound regulators of complement activity on human uveal melanoma cells

Tumor cells are protected from antibody-dependent complement-mediated lysis by membrane-bound regulators of complement activation (m-RCA). m-RCA are expressed on uveal melanoma cells. We determined whether cytokine treatment affects expression of m-RCA on these cells in vitro. m-RCA expression on uveal melanoma cell lines was studied by flow cytometry, using monoclonal antibodies directed against CD46, CD55, and CD59. Cytokines studied were interferon-alpha (IFN-alpha), IFN-gamma, interleukin-1B (IL-1B), IL-12, and tumor necrosis factor-alpha (TNF-alpha). All three m-RCA were expressed on the uveal melanoma cell lines (CD59>CD46>CD55), although in variable amounts. With a few exceptions, the cytokines had no effect on m-RCA expression. CD55 expression was not influenced by any of the cytokines. IFN-gamma downregulated expression of CD46 on one cell line (p < 0.01). TNF-alpha upregulated CD59 expression on two of the five cell lines (p < 0.012 and p < 0.001, respectively), which effect was dose dependent. IFN-alpha, IFN-gamma, IL1-beta, IL12, and TNF-alpha had limited effects on m-RCA expression on uveal melanoma cells in vitro.     

Identification of nuclear matrix protein alterations associated with renal cell carcinoma

PURPOSE: Neoplastic transformation, including renal cell carcinoma (RCC), is always accompanied by changes in nuclear morphology. Nuclear grading of RCC is based on characteristic alterations in nuclear shape, size, area and other morphologic parameters. The nuclear matrix, which forms the skeleton of the nucleus, determines nuclear morphology. Alterations in nuclear matrix protein (NMP) composition specific to tissue and cancer type have been described in a variety of human cancers. We conducted a study to analyze the nuclear matrix protein composition of renal cell carcinoma and compare it to that of normal renal tissue and renal cell carcinoma cells grown in culture. MATERIALS AND METHODS: We analyzed the nuclear matrix protein composition of RCC tumor tissue and that of normal kidney tissue obtained from seventeen patients undergoing radical nephrectomy for RCC. We also analyzed the NMP composition of two renal cancer cell lines (A-498 and 769-P). RESULTS: We were able to identify five different and unique NMPs which were present only in the human RCC tumor samples and were absent in all normal kidney tissue. One NMP was found specifically in the normal kidney tissue. All five RCC specific NMPs were also identified in the nuclear matrix of the two cell lines analyzed. CONCLUSIONS: Five nuclear matrix proteins specific and unique to RCC were identified. These NMPs are different from those previously identified in other tissues and neoplasms. The RCC specific NMPs identified in this study can potentially be used as diagnostic markers for renal cell carcinoma and for therapeutic tumor targeting.     

Fas/APO-1 (CD95)-mediated apoptosis is activated by interferon-gamma and interferon- in interleukin-6 (IL-6)-dependent and IL-6-independent multiple myeloma cell lines

A poor response to Fas-induced apoptosis is evident in some multiple myeloma (MM) cell lines and primary cells. In this study, we have examined the possibility to increase the sensitivity to Fas-induced apoptosis by pretreatment of MM cells with interferon-gamma (IFN-gamma) or interferon-alpha (IFN-alpha). Both IFN-gamma and IFN-alpha markedly increased the Fas-induced apoptosis in all cell lines tested (U-266-1970, U-266-1984, and U-1958). In the U-266-1970 and U-1958 cell lines, pretreatment with either IFN-gamma or IFN-alpha also inhibited proliferation in a dose-dependent manner. In contrast, IFN-gamma activation of the Fas death pathway in the U-266-1984 cells was not accompanied by growth inhibition. Incubation with the IFNs increased the Fas antigen expression in one of three cell lines but did not alter the expression of Bcl-2 or Bax. The IFNs are important regulators of growth and survival in MM cells. Our results suggest that activation of Fas-mediated apoptosis is a novel mechanism by which the IFNs exert inhibitory effects on MM cells.     

Synergistic interaction between cisplatin and gemcitabine in vitro

2',2'-Difluorodeoxycytidine (dFdC; gemcitabine) is a new antineoplastic agent that is active against ovarian carcinoma, non-small-cell lung carcinoma, and head and neck squamous cell carcinoma. cis-diamminedichloroplatinum (CDDP; cisplatin) is used commonly for the treatment of these tumors. Because the two drugs have mechanisms of action that might be complementary, we investigated a possible synergism between dFdC and CDDP on growth inhibition. The combination was tested in the human ovarian carcinoma cell line A2780, its CDDP-resistant variant ADDP and its dFdC-resistant variant AG6000, the human head and neck squamous cell carcinoma cell line UMSCC-22B, and the murine colon carcinoma cell line C26-10. The cells were exposed to dFdC and CDDP as single agents and to combinations in a molar ratio of 1:500 for 1, 4, 24, and 72 h with a total culture time of 72 h. Synergy was evaluated using the multiple drug effect analysis. In A2780 and ADDP cells, simultaneous exposure to the drugs for 24 and 72 h resulted in synergism, but shorter exposure times were antagonistic. No synergism was found in the UMSCC-22B and C26-10 cell lines at prolonged simultaneous exposure. However, a preincubation with CDDP for 4 h followed by a dFdC incubation for 1, 4, 24, and 72 h was synergistic in all cell lines except C26-10 cells. A 4-h preincubation with dFdC followed by an incubation with the combination for 20 and 68 h was synergistic in all cell lines. Initial studies of the mechanism of interaction concentrated on the effect of CDDP on dFdCTP accumulation and DNA strand break formation. In all cell lines, CDDP failed to increase dFdCTP accumulation at 4- or 24-h exposure to dFdC; in two cell lines, CDDP even tended to decrease dFdCTP accumulation. Neither dFdC nor CDDP caused more than 25% double strand break formation, whereas in the combination, CDDP even tended to decrease this type of DNA damage. The synergistic interaction between the two drugs is possibly the result of dFdC incorporation into DNA and/or CDDP-DNA adduct formation, which may be affected by each other.     

IFN-alpha 2B enhances Th1 cytokine responses in bladder cancer patients receiving Mycobacterium bovis bacillus Calmette-Guérin immunotherapy

Combination therapy with intravesical bacillus Calmette-Guérin (BCG) plus IFN-alpha for superficial bladder cancer has been demonstrated to be more effective than either single agent alone in animal studies and of suggested greater efficacy in clinical studies. However, the mechanism by which IFN-alpha enhances BCG-mediated antitumor activity is poorly understood. Using PBMCs from bladder cancer patients, IFN-alpha was found to substantially enhance the efficacy of BCG to induce IFN-gamma production. Among 34 patients tested, 80% showed >4-fold increase. This effect of IFN-alpha was observed in both initial and memory responses to BCG. In addition, IFN-alpha up-regulated BCG-induced IL-12 and TNF-alpha and down-regulated BCG-induced IL-10. Neutralizing endogenous IL-10 or adding exogenous IL-12 provided further synergy for IFN-gamma production. In clinical practice, intravesical IFN-alpha 2B (50 million units (MU)/dose) was observed to accelerate urinary IFN-gamma production to low-dose BCG (one-tenth or one-third of a full dose) in patients treated with combination therapy compared with BCG alone. These results suggest that IFN-alpha is a potent BCG enhancer that polarizes the BCG-induced immune response toward the cellular immune pathway by promoting Th1 cytokine expression and reducing Th2 cytokine expression. This study provides an immunological basis for future rational use of IFN-alpha in conjunction with intravesical BCG for bladder cancer immunotherapy.     

Decreased resistance to gemcitabine (2',2'-difluorodeoxycitidine) of cytosine arabinoside-resistant myeloblastic murine and rat leukemia cell lines: role of altered activity and substrate specificity of deoxycytidine kinase

We determined the potential activity of 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in 1-beta-D-arabinofuranosylcytidine (ara-C)-sensitive and-resistant leukemia cell lines. Both drugs are phosphorylated by deoxycytidine kinase (dCK); the triphosphates, dFdCTP and ara-CTP, respectively, are incorporated into DNA. In the murine leukemia cell line L1210, induction of resistance to ara-C resulted in the 2200-fold resistant subline L4A6. The Brown Norway rat myelocytic leukemia ara-C-sensitive cell line (BCLO) was >300-fold more sensitive to ara-C than its variant Bara-C. In L1210 cells, gemcitabine was 8-fold more active than ara-C; in L4A6, BCLO, and Bara-C cells, gemcitabine was 16-, 28-, and more than 3-fold more active than ara-C, respectively. A partial explanation for these differences may be the higher dCK activity in the parental cell lines L1210 and BCLO with gemcitabine compared to ara-C as a substrate. DCK activity was not or hardly detectable in the resistant L4A6 and Bara-C cell. In the rat leukemia cell lines, deoxycytidine (dCyd) phosphorylation activity showed an aberrant pattern, since the activity with dCyd was 1.5-fold higher in the Bara-C cell line compared with BCLO, possibly due to thymidine kinase 2. The wild-type L1210 cells accumulated at least 3-fold more ara-CTP and dFdCTP than the rat leukemia cell line BCLO. The ara-C-resistant variants L4A6 and Bara-C did not accumulate dFdCTP or ara-CTP. In conclusion, gemcitabine was more active than ara-C in all leukemia cell lines tested. The sensitivity of the wild-type cell lines correlates with the accumulation of dFdCTP and ara-CTP, but is independent of dCK. However, both resistant variants had decreased dCK activities, but were relatively more sensitive to dFdC than to ara-C.     

The role of Stat4 in species-specific regulation of Th cell development by type I IFNs

Type I IFNs (IFN-alpha/beta), in addition to IL-12, have been shown to play an important role in the differentiation of human, but not mouse, Th cells. We show here that IFN-alpha/beta act directly on human T cells to drive Th1 development, bypassing the need for IL-12-induced signaling, whereas IFN-alpha cannot substitute IL-12 for mouse Th1 development. The molecular basis for this species specificity is that IFN-alpha/beta activate Stat4 in differentiating human, but not mouse, Th cells. Unlike IL-12, which acts only on Th1 cells, IFN-alpha/beta can activate Stat4 not only in human Th1, but also in Th2 cells. However, restimulation of human Th2 lines and clones in the presence of IFN-alpha does not induce the production of IFN-gamma. These results suggest that activation of Stat4, which is necessary for the differentiation of naive T cells into polarized Th1 cells, is not sufficient to induce phenotype reversal of human Th2 cells.     

Reversal of left ventricular hypertrophy after the use of benazepril

gamma-Interferon (IFN-gamma) was detected by ELISA assay in ascitic fluid from a number of ovarian cancer patients. To study its clinical significance, the effect of IFN-gamma on the metastatic potential of a mouse mammary adenocarcinoma, MA-891, was explored. Pretreatment of the tumor cells in vitro for 48hr with recombinant INF-gamma significantly increased the number of lung tumor nodules after i. v. or s. c. inoculation into (TA2 x 615) F1 mice. In contrast, when recombinant IFN-alpha pretreated MA-891 cells were likewise injected into mice significant decrease in metastatic potential was seen. The study in vitro indicated that pretreatment of the tumor cells with IFN-gamma but not with IFN alpha resulted in a decrease in susceptibility to NK cell cytotoxicity. In as much as both IFN-alpha and IFN-gamma can induce MHC class I expression on target cells. The increase in metastatic potential of IFN-gamma-treated tumor cells can be explained only partially on the basis of their reduced NK cells susceptibility.     

Serum interleukin 4 and interleukin 10 levels in patients with chronic hepatitis C virus infection

The antiproliferative effect of 5-fluorouracil (5-FU) in colon cancer can be enhanced by interferons (IFN-alpha and IFN-gamma). The mechanisms by which IFNs modulate 5-FU activity are not completely elucidated. IFN-alpha may elevate the levels of the active 5-FU metabolite 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) in the cell, possibly leading to increased inhibition of the target enzyme thymidylate synthase (TS), which might enhance DNA damage. It has been shown that IFN-gamma can prevent 5-FU induced overexpression of TS. We studied IFN modulation in three colon cancer cell lines (SW948, WiDr, human; C26-10, murine) and the sublines WiDr/F and C26-10/F, which were adapted to low folate levels. A 1.5-fold increase in 5-FU sensitivity was observed in C26-10 and C26-10/F (by murine IFN-alpha, beta); in SW948, WiDr and WiDr/F (by human IFN-gamma) and in SW948 and WiDr/ F (by human IFN-alpha). In none of the cell lines did human IFN-alpha, IFN-gamma or murine IFN-alpha, beta increase FdUMP levels after exposure to 5-FU. TS activity, indirectly measured by incorporation of [6-3H]-deoxyuridine into DNA, was inhibited by 5-FU, but the IFNs did not enhance inhibition. DNA damage was measured as a drug-induced decrease of double-stranded (dss) DNA compared to control cells. After 5-FU exposure, dss DNA decreased to 60-75% in WiDr, WiDr/F and SW948 cells. Human IFN-alpha alone caused minimal DNA damage (95% dss DNA), but increased 5-FU-induced effects to 35-50% dss DNA. IFN-gamma did not cause DNA damage and did not enhance 5-FU-mediated DNA damage. Expression of TS protein, analysed by ELISA, was increased after 5-FU exposure of SW948 cells, but this increase was not affected by addition of either IFN-alpha or IFN-gamma. It is concluded that one of the mechanisms involved in modulation of 5-FU activity is the effect of IFN-alpha on 5-FU-mediated DNA damage, but for IFN-gamma no mechanism of action was found.     

Tetrapeptide DEVD-aldehyde or YVAD-chloromethylketone inhibits Fas/Apo-1(CD95)-mediated apoptosis in renal-cell-cancer cells

The apoptotic machinery has been intensively investigated, and interleukin-1-beta-converting enzyme (ICE) and its homologs directly mediate apoptosis by means of their unique protease activity. Fas/Apo1 (CD95), a member of the TNF-receptor family, mediates apoptosis by binding to its ligand, which is mainly expressed on lymphocytes. Here, we investigated the expression and function of both molecules in renal-cell cancer (RCC). The expression of Fas was examined in 6 RCC cell lines by immunoblotting and all of them expressed Fas. ICE and CPP32/YAMA were also identified among the cell lines. We earlier examined ACHN cells expressing low levels of BCL-2, as well as KRC/Y cells with high levels of BCL-2. Here, we found that the anti-Fas monoclonal antibody, CH-11, induced apoptosis in a dose-dependent fashion more remarkably in ACHN cells. Pre-incubation with the tetrapeptide YVAD-chloromethyl-ketone or DEVD-aldehyde inhibited Fas-mediated apoptosis. These findings suggest that, in RCC, apoptosis is induced by lymphocytes bearing Fas-L, and that it is achieved through the proteolytic action of CPP32/YAMA and/or ICE, or another member of the ICE/ced-3 protease family.     

Effects of interferon-alpha and gamma on development of LAK activity from mononuclear cells in breast cancer patients

We examined the effect of recombinant IFN-alpha and IFN-gamma on induction of LAK cells from peripheral blood mononuclear cells (PBMNCs) in 7 pre-operative breast cancer patients and 4 healthy volunteers. Significant LAK activity was developed from PBMNCs of pre-operative breast cancer patients and healthy volunteers after incubation for 4 days with IL-2 (presence of IL-2 vs. absence of IL-2). Incubation of PBMNCs of pre-operative breast cancer patients with 1000 U/ml of IFN-alpha for 4 days suppressed the LAK activity significantly (P < 0.05). By contrast, incubation of PBMNCs of pre-operative patients with 1000 U/ml of IFN-gamma for 4 days increased the LAK activity significantly (P < 0.05). Significant cytotoxicity against MCF-7 cells (estrogen receptor positive human breast cancer cell line) was developed from PBMNCs of pre-operative breast cancer patients at 20:1 and 40:1 E/T ratios after incubation for 4 days with IL-2 (absence of IL-2 vs. 20:1 or 40:1, P < 0.05, P < 0.05), whereas PBMNCs of healthy volunteers did not. Stimulation of LAK cells with IFN-gamma produced a significant augmentation of cytotoxic activity against MCF-7 (P < 0.05), while IFN-alpha suppressed the cytotoxicity significantly (P < 0.05). These findings suggested that combined stimulation by IFN-gamma and IL-2 might be a reasonable treatment for breast cancer patients.