Search

In general, chemotherapeutic effects were low for non-small cell lung cancer (NSCLC) in the lung tumor. We examined the accumulation and antitumor effects of hybrid liposomes (HL-23) composed of phospholipid (L-α-dimyristoylphosphatidylcholine: DMPC) and PEG surfactant [polyoxyethylene(23)dodecyl ether: C₁₂(EO)₂₃] on NSCLC cells in vitro. Accumulation of HL-23 including a fluorescence probe [1-Palmitoyl-2-[12(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-Glycero-3-Phosphocholine: NBDPC] was observed for NSCLC cells using a confocal laser microscope, but no accumulation of HL-23 in normal lung cells was observed. Furthermore, inhibitory effects of HL-23 on the growth of NSCLC cells were obtained on the basis of a WST-1 assay. It was also clarified that HL-23 induced apoptosis for NSCLC cells on the basis of Annexin-V binding and TUNEL assay. These results suggest that HL-23 could be applied in effective chemotherapies for NSCLC.

Constitutive activation of the extracellular signal-regulated kinase (ERK) and/or phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathways is associated with neoplastic transformation of a variety of human tumor cells. Treatment of such tumor cells with agents that specifically inhibit the activity of mitogen-activated protein kinase/ERK kinase (MEK) or PI3K completely suppresses their proliferation. However, treatment of cells with these inhibitors leads to only a modest increase in apoptotic cell death. Efficient induction of apoptosis is essential for the development of effective cancer chemotherapy. Therefore, we have examined whether specific blockade of these two signaling pathways affects the efficacy of the induction of apoptosis by various anti-cancer agents in tumor cells. We found that MEK inhibitors markedly enhance the efficacy of histone deacetylase inhibitors to induce the generation of reactive oxygen species and apoptosis. This effect was only observed in tumor cells in which the ERK pathway was constitutively activated. Furthermore, we found that PI3K inhibitors selectively and markedly enhanced the efficacy of the induction of apoptosis by doxorubicin. This latter effect was only detected in tumor cells in which the PI3K/Akt pathway was constitutively activated and in which the p53 pathway was functional. These combination treatments provide an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway or PI3K/Akt pathway is constitutively activated. This review gives an overview of the development of new targeted molecular strategies for cancer therapy based on the suppression of the activity of oncogenic pathways involved in the proliferation or survival of tumor cells.

We evaluated the usefulness of fusion vaccine prepared from IL-2-gene-transduced splenic dendritic cells (DCs) and fibrosarcoma tumor cells (QRsP) in treating of lung metastasis. The IL-2 or LacZ gene was transferred into spleen-derived DCs using an adenoviral vector. Irradiated QRsP tumor cells were fused with IL-2 gene transduced DCs (fusion/IL-2) or LacZ gene transduced DCs (fusion/LacZ) by polyethyleneglycol. These fusion cells expressed major histocompatibility complex (MHC) class I and MHC class II, CD86, CD11c and CD8alpha. IFN-gamma and cytotoxic T lymphocyte (CTL) activity of splenic lymphocytes in mice vaccinated with fusion cells increased significantly as compared with those of DC or tumor cells vaccinated mice. CTL levels in fusion/IL-2-vaccinated mice were higher than that in fusion/LacZ-vaccinated mice. The number of lung metastasis in the fusion/IL-2 or fusion/LacZ-vaccineatd mice was significantly lower than that in mice vaccinated with DCs, tumor or PBS. The introduction of the IL-2 gene into fusion cells produced more potent therapeutic effects. Our results suggest that the fusion cells prepared from IL-2 gene transduced spleen derived DCs and tumor cells have the ability to induce therapeutic effect against lung metastasis.

The inhibitory effects of hybrid liposomes composed of lipids having a variety of head groups (zwitterionic L-alpha-dimyristoylphosphatidylcholine (DMPC), anionic L-alpha-dimyristoylphosphatidylglycerol (DMPG), and cationic 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP)) and polyoxyethylene (10) dodecyl ether (C12(EO)10) on the growth of tumor cells (human lung carcinoma (RERF-LC-OK), human hepatoma (Hep-G2), human stomach tumor (GT3TKB)) in vitro were examined. The hybrid liposomes of DMPC/10 mol% C12(EO)10 and DMPG/10 mol% C12(EO)10 were fairly more effective for inhibiting the growth of all tumor cells employed in this study as compared with liposomes (DMPC and DMPG) or micelles (C12(EO)10). Especially, it is attractive that the highly specific inhibitory effect of the hybrid liposomes of DMPG/10 mol% C12(EO)10 without any antitumor drugs on the growth of RERF-LC-OK and GT3TKB was obtained for the first time.

A weakly tumorigenic cell clone (QR-32) derived from a murine fibrosarcoma (BMT-11) grew lethally in 6 out of 10 syngeneic C57BL/6 mice after co-implantation with gelatin sponge. All six cell lines (QRsP) established from the arising tumors from QR-32 had enhanced tumorigenicity and/or pulmonary metastatic ability in vivo, indicating that those QRsP cell lines acquired progressed phenotypes as compared with those of QR-32 cells. In contrast, the frequency of tumor progression was suppressed to 50% (3/6) in the cell lines (QRsP/PSK) established from those arising in the mice treated with an immunopotentiating protein-bound polysaccharide, PSK. The enhanced metastatic ability was accompanied by enhanced expressions of a tumor-associated transcription factor, E1AF and by increased production of matrix metalloproteinase (MMP) in five lines of QRsP and two lines of QRsP/PSK. It was found that administration of PSK augmented the production of an antioxidative enzyme, manganese superoxide dismutase (Mn-SOD), in the tumor tissues co-implanted with gelatin sponge. PSK administration also brought about up-regulation of interferon-gamma (IFN-gamma)-expression and down-regulation of transforming growth factor-beta (TGF-beta)-expression in the tumor tissues, which were examined by RT-PCR on day 7, 14 and 21 after the co-implantation. Other inflammatory cytokines such as interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) were expressed equally both in PSK-treated and untreated tumor tissues. In vitro experiments proved that although IFN-gamma did not increase the production of Mn-SOD by itself, concomitant treatment with both IFN-gamma and TNF-alpha enhanced the Mn-SOD-production in QR-32 cells greatly. On the contrary, TGF-beta treatment lowered the Mn-SOD level in QR-32 cells. PSK-treatment did not induce Mn-SOD in cultured QR-32 cells directly. These results indicated that PSK inhibits the malignant progression of QR-32 cells promoted by co-implantation with gelatin sponge, most possibly through elevating the Mn-SOD level in QR-32 cells via modulation of the production of inflammatory cytokines, that is, increasing IFN-gamma and decreasing TGF-beta at the site of tumor growth.

Trichostatins and trapoxins, structurally unrelated microbial metabolites, are specific inhibitors of histone deacetylases. Radicicol inhibits Src family protein-tyrosine kinases. Recently, these agents were found to induce morphological reversion and enhanced expression of gelsolin, an actin regulatory protein, in a variety of transformed cells. Microinjection of an anti-gelsolin antibody that neutralizes the gelsolin function caused inhibition of the morphological change, suggesting that gelsolin expression is associated with the suppression of transformation.

Nitrosoureas are antitumor alkylating agents widely used in the chemotherapy of malignant brain tumors. However, the effectiveness of adjuvant nitrosourea chemotherapy has proved inadequate, failing to provide any significant prolongation of survival time. One of the reasons for the poor results is a drug resistance system in the form of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). O6-alkylguanine derivatives are well known to be inhibitors of MGMT, and inactivation of MGMT by these derivatives leads to increased tumor cell sensitivity to nitrosoureas. In this study, the authors tested the ability of O6-benzylguanine, O6-(4-, 3- and 2-fluorobenzyl) guanines, O6-(4-, 3- and 2-trifluoromethylbenzyl) guanines, O6-(4-, 3- and 2-pyridylmethyl) guanines and O6-(2- and 1-naphthylmethyl) guanines to reduce MGMT activity in SF-188 cell-free extract by using [3H] methylated substrate DNA and analyzed their enhancing effect on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl) -3-nitrosourea hydrochloride (ACNU) by using a calorimetric cytotoxicity assay. The MGMT activity in the SF-188 cell-free extract was 944 +/- 43 fmol/mg protein (Mean +/- SD, n = 5). O6-(4- and 3-fluorobenzyl) guanines were found to be more effective in inactivating MGMT than O6-benzylguanine. O6-(4-trifluoromethylbenzyl) guanine considerably reduced MGMT activity as did O6-benzylguanine. O6-(3-trifluoromethylbenzyl) guanine, O6-(4- and 3-pyridylmethyl) guanines, and O6-(2-naphthylmethyl) guanine were intermediately effective, but O6-(2-fluorobenzyl) guanine, O6-(2-trifluoromethylbenzyl) guanine and O6-(1-naphthylmethyl) guanine were less effective. ACNU cytotoxicity in SF-188 cells was strongly enhanced by pretreatment with O6-(4- and 3-fluorobenzyl) guanines and O6-(4-trifluoromethylbenzyl) guanine and moderately enhanced by O6-(3- trifluoromethylbenzyl) guanine and O6-(4- and 3-pyridylmethyl) guanines, but not enhanced by O6-(2-fluorobenzyl) guanine, O6-(2-trifluoromethylbenzyl) guanine and O6-(1-naphthylmethyl) guanine. The test compounds were not cytotoxic at concentrations between 0.5 and 5.0 microM. The enhancing effects on ACNU cytotoxicity were consistent with the inhibition of MGMT activity after two-hour pretreatment with O6-arylmethylguanine derivatives. These results indicate that the 2-position of the O6-benzyl group plays an important role in the inactivation of the MGMT activity and the potentiation of ACNU cytotoxicity.

Potentially antigenic tumors often escape from the immune surveillance system by producing immunosuppressive factors. It has previously been reported that a progressor-type murine fibrosarcoma culture line, QRpP cells, produced high levels of PGE2 compared with a regressor-type tumor line, QR-32 cells, and that QRpP cells progressively grew in syngeneic C57BL/6 mice. In order to improve suppressed immunogenicity of QRpP cells with the high levels of PGE2, the author transfected QRpP cells with an effective expression vector containing cDNA for IL-2, IFN-gamma and TNF-alpha. These transfected clones expressed mRNA for IL-2, IFN-gamma and TNF-alpha, respectively, and produced high levels of cytokines in their culture supernatants. Consequentry, QRpP cells transfected with IL-2 (QRpP-IL-2), IFN-gamma (QRpP-IFN-gamma), IL-2 and IFN-gamma (QRpP-IL-2/IFN-gamma), TNF-alpha (QRpP-TNF-alpha) lowered in tumorigenicity. In particular, the mice that had rejected the implantation of QRpP-IL-2 clone also rejected implantation of the parental QRpP cells. The activity of pre-cytotoxic T cells (pre-CTLs) obtained from the mice implanted with QRpP-IL-2 clone was higher than that of the mice implanted with other transfectants. These results suggested that transfection with the gene for IL-2 is an effective strategy against the producing immunosuppressive factors such as PGE2.

We have detected both germ-line and somatic p53 mutations in lymphocytes, cell lines and tumor tissues using a functional analysis of p53 tumor suppressor gene based on yeast transcription assay. Through our screening projects of the p53 gene, a number of missense p53 mutations were identified as loss-of-function mutations. This method, previously termed FASAY, is rapid, sensitive, less-expensive and can be automated for screening both somatic and germ-line p53 mutations.

The mode of cell death in cells undergoing mitotic death after irradiation was studied in twenty cancer cell lines of human gastrointestinal origin. Apoptotic cells were observed in all cancer cell lines after irradiation, whereas two fibroblast strains were quite low in apoptosis frequency. The advent of apoptosis depended on the radiation doses and incubation time. Detailed analysis of one of the carcinoma lines, SH101-P4, revealed that G2 phase arrest was maximum at 12 h postirradiation. The cells began to escape G2 arrest by 24 h. Apoptotic cells began to increase at 12 h postirradiation and became maximal from 72 to 96 h. Apoptosis developed in the G1 phase of the cell cycle subsequent to the irradiation. Moreover, the rate of apoptosis of the cells by irradiation did not correlate with p53 gene abnormalities. These results suggest that apoptosis is one of the modes of mitotic death after irradiation.

Anti-cancer effect of anthracyclines are well established. In vitro inhibitory effects of a new anthracyclin-derivative drug, menogaril (TUT-7) which is developed for oral administration, on three human T-cell leukemia virus type-I (HTLV-I) infected T-cell lines, a HTLV-I non-infected T cell line, and fresh tumor cells from four adult T-cell leukemia (ATL) cases are evaluated and compared to those of doxorubicin. The inhibitory effects of TUT-7 is almost as much as those of doxorubicin. Both TUT-7 and doxorubicin induced apoptosis to a HTLV-I infected cell line and inhibited proliferation of fresh tumor cells from ATL of chronic, acute and lymphoma type in a dose dependent manner. We have already reported that skin eruption of ATL patient improved by oral administration of TUT-7. This new anthracyclin-derivative would be useful in treating ATL patients even in out-patients clinic keeping a good quality of life.

This study investigated the role of activated macrophages (M phi) in nitric oxide (NO) production and the tumoricidal effect of NO on glioma cells. Induced peritoneal M phi were prepared 6 days following the injection of thioglycollate broth into C3H/He N (H-2 kappa) mice. M phi were activated in vitro recombinant human interferon-gamma (IFN gamma) and lipopolysaccharide (LPS) into the culture medium of the elicited M phi. Two kinds of murine malignant glioma cell lines, RSV-M glioma (H-2 kappa) and VM-glioma (H-2b) were used as targets. P815 mastocytoma cells (H-2d) were used as a control target, since they are insensitive to tumor necrosis factor-alpha, but susceptible to NO derived from M phi. L-arginine-depleted medium was used to inhibit NO-mediated cytocidal activity against tumor cells. Cytotoxicity was assayed at various effector-to-target ratios using an admixture of M phi and 1.5 x 10(4) 125I-labeled target cells 48 hours following co-culture. NO was measured in culture medium using Griess reagent, and the concentration of NO was expressed as mu mol/ml NaNO2. Peritoneal M phi induced only 10% and 15% lysis of RSV-M glioma and VM glioma cells, respective, and LPS augmented this killing activity of M phi to a maximum of 1.2 to 1.4 fold in a dose-dependent manner with dosages from 1 to 50 ng/ml. LPS demonstrated a synergistic action on M phi-mediated cytotoxicity 4 hours following pretreatment with IFN gamma. Alternatively, low doses of IFN gamma alone had no enhancing effect on M phi tumoricidal activity.(ABSTRACT TRUNCATED AT 250 WORDS)

RBT-K5 cell line was established from a male Wistar rat bladder tumor induced with N-butyl-N-(4-hydroxybutyl) nitrosamine. RBT-K5 (ADM res.) cell line, adriamycin (ADM) resistant, was also established from RBT-K5 cells by stepwise escalation of ADM concentrations in vitro. In this study, we have examined the effect of verapamil (VR) on intracellular ADM concentration in these cell lines. The viability of RBT-K5 cells was significantly lower than that of RBT-K5 (ADM res.) at the concentration from 0.5 to 4 micrograms/ml of ADM. The cytotoxic effect of ADM was enhanced by the addition of VR in the both cell lines. The intracellular ADM concentrations of RBT-K5 and RBT-K5 (ADM res.) were increased from 607.7 +/- 74.1 ng/10(6) cells to 1152.1 +/- 187.6 ng/10(6) cells (1.9-fold) and from 185.1 +/- 36.5 ng/10(6) cells to 751.5 +/- 258.4 ng/10(6) cells (4.1-fold), respectively, 24 hours after incubation in the presence of 10 micrograms/ml of VR. Furthermore, the intracellular ADM concentration was maintained at high-level in combination with VR even after the removal of ADM from culture medium. These results suggest that the combination therapy with VR is more effective against ADM-resistant than ADM-sensitive bladder tumor.

Various anticancer agents have been known to induce apoptosis in certain types of human tumor cells. The fact that a variety of agents, which attack different cellular targets, induce common apoptotic cell death suggests that the nature of initial damage is not directly involved in apoptosis. The mechanism by which a damage leads to apoptosis is not known. However, modulation of this process may affect the outcome of anticancer drug treatment. This article briefly reviewed the studies of endogenous as well as exogenous factors which modulate apoptosis, and then described the characteristics of cell death induced by alkylating agents. O6-Alkylguanine, a major cytotoxic DNA damage produced by simple alkylating agents, can be repaired by the cellular enzyme O6-methylguanine-DNA methyltransferase (MGMT). About one-fifth of human tumor cell strains lack the MGMT activity and termed as Mer- cells. Mer- cells are hypersensitive to alkylating agents like chloroethyl nitrosoureas (CNUs), compared with repair-proficient Mer+ cells. It is suggested that identification of a factor which suppresses the MGMT gene expression in CNU-resistant Mer+ cells, may enable us to convert these Mer+ cells to Mer- phenotype, thus resulting in much higher sensitivity of Mer+ cells to CNUs.

I studied alterations in the tumorigenicity and immunogenicity of cultured rat fibrosarcoma clone in different growth environments. When the fetal calf serum concentration in a medium was lowered from 10% to 1%, the tumorigenicity was diminished while the immunogenicity was enhanced, accompanied by a prolongation of the in vitro doubling time in a reversible manner. These phenomena were not associated with the quantities of the tumor-associated antigen and/or the rat major histocompatibility complex, but were associated with the appearance of a unique antigen (s), which consist of glycoprotein and exist as a crypt-antigen (s). Our observations indicate that the in vitro growth environment modifies the surface of tumor cells and causes a reduction in their tumorigenicity and an enhancement of their immunogenicity.

Image analysis of the nuclear morphology was performed on propidium iodide-stained isolated cells from 9 adenomas and 9 cancers of the colorectum. We analyzed DNA content, seven nuclear geometric features and four shape factors of tumor cells using an image cytometry system that has been developed in our laboratory. Nuclear breadth and the degree of contour irregularity of cancer cells were found to be significantly different from those of adenoma cells. The coefficients of variation (CV) of some nuclear features and the degree of contour irregularity were increased in cancer cells, while CV of the degree of circularity was increased in adenoma cells. Moreover, nuclear in G2 phase were found to be larger and more round than those in G1 phase. These results suggest that cell-cycle-related analysis of nuclear features would be a valid means to assess the nuclear morphology of colorectal tumors.

We examined factors promoting malignant progression using a weakly malignant variant cell line, ER-1, derived from c-SST-2, a rat mammary carcinoma. ER-1 cells were converted to a highly malignant phenotype (highly tumorigenic, metastatic, invasive in vitro) by the in vitro/in vivo interaction with host cells reactive to foreign body. Epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) produced by host reactive cells, transiently enhanced the tumorigenicity and in vitro invasiveness of ER-1 cells into an endothelial cell monolayer. The host reactive cells also produced oxygen radicals and induced mutations in ER-1 cells. It is speculated that mutations induced by host reactive cells cause cellular diversification, including the emergence of highly malignant variant cells whose growth is selectively promoted by growth factors such as EGF and TGF-beta.

Effects of pretreatment with N-methyl-N-nitrosourea (MNU) or streptozotocin (STZ) on cytotoxicity and induction of sister chromatid exchanges (SCE) in human and rodent brain tumor cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 1-(2-chloroethyl)-1-nitrosourea (CNU) were investigated. 9L-2 and SF-188 cells were more resistant to the cytotoxic effects of chloroethylnitrosourea than 9L and SF-126 cells. SF-295 cells were more resistant to the cytotoxic effects than SF-126, but more sensitive than SF-188 cells. Pretreatment of 9L-2 cells with MNU or STZ resulted in a dose-dependent increase in cytotoxicity and SCE induction by BCNU. Treatment with 1mM MNU or 1.5mM STZ completely reversed the cellular resistance of 9L-2 cells to BCNU but did not potentiate either cytotoxicity or SCE induction in 9L cells. Pretreatment of SF-188 and SF-295 cells with MNU or STZ resulted in a dose-dependent increase in cytotoxicity and SCE induction by CNU. Treatment with 500 microM MNU or 1.5mM STZ for SF-188 cells, and with 250 microM MNU or 1.5mM STZ for SF-295 cells completely reversed the cellular resistance to CNU. These results are consistent with the hypothesis that pretreatment with MNU or STZ inhibits O6-alkylguanine-DNA-alkyltransferase (O6-AT) and inhibition of the enzyme allows the formation of DNA interstrand cross-links resulting in increase in cytotoxicity and induction of SCE in resistant cells treated with chloroethylnitrosourea. In this regard, O6-AT plays an important role in determining the cytotoxicity and induction of SCE by chloroethylnitrosourea in both rodent and human brain tumor cells.

Enrichment of complex type N-linked sugar chains containing the Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----4GlcNAc beta 1----2)Man group was found to be the structural background of Warren-Glick phenomenon. This alteration is induced by enhanced expression of N-acetylglucosaminyltransferase V in malignant cells. The phenomenon is positively correlated with the tumorigenicity and the potency of blood-borne metastasis of tumor cells. Further investigation of the surface sugar chains of lectin mutants of mouse B16 melanoma revealed that the occurrence of sialylated tetraantennary sugar chains is essential for the metastasis of tumor cells.

Tumor cell lines in culture that can grow in protein-free medium are suitable for studying the mechanism of autonomous growth of tumor cells. We have studied the mechanism of autonomous growth of three cell lines which were established from human solid tumors. These cell lines produce basic fibroblast growth factor (bFGF), but it does not seem to act as an autocrine growth factor. bFGF produced by these cells is accumulated in the nuclei and may act as an autointrafactor for autonomous growth of these cells.

Ehrlich ascites tumor cells (EAT) could not proliferate in a basal medium (BM). L-Cysteine (L-CYS) added to BM in a concentration of 0.5 to 2 mM could promote the growth of EAT. At an unusual concentration of L-CYS (1 mM), the growth-rate was 233% (n = 50) at 72 hr after EAT transplantation. A similar growth-rate was obtained when EAT was transplanted 24 hr after addition of L-CYS to BM, although the free SH content in L-CYS-added BM at this time decreased to the level of that in BM. S-Methyl- and S-benzyl-L-cysteine showed no growth-promoting effect. The metabolic products of L-CYS (L-cysteine sulfinic acid, hypotaurine and taurine) showed a lower effect or none, but L-cystine showed the same activity as L-CYS. The EAT cultured in L-CYS-added BM was found to have an increased intracellular free SH and unchanged protein SH. These findings suggest that L-CYS added to BM is changed in a non-SH compound (possibly L-cystine) that is taken up into the cells and increases the intracellular free SH, resulting in the cell growth promotion.

In order to analyse a virus-associated antigen which is expressed on Friend virus-infected rat tumor cells and induces strong resistance to their transplantability. I made monoclonal antibodies against it by using spleen cells of the syngeneic rats in whom the Friend virus-associated tumor cells spontaneously regressed. As a result, I was able to obtain a monoclonal antibody, named TF 1, to react specifically to the infection of Friend virus and found out that TF1 antibody reacted to gp 70, a product of the envelope gene of Friend virus, on Friend virus-infected rat tumor cells. And also I found out that B16BL6 cells, which are mouse melanoma cells and are not infected with Friend virus, were positive to TF1 antibody and made it clear that the melanoma antigen which reacted to TF 1 antibody was not gp 70, but a protein of 80-85 KD. Immunizing therefore with gp 70 cross-reacting with the melanoma antigen, I was able to observe that resistance to the transplantability of B16BL6 cells was induced. The result suggests that the melanoma antigen played a role as a tumor rejection antigen. I thus consider that gp 70 is able to induce resistance to transplantability of B16BL6 cells in mice and affects spontaneous regression of Friend virus-infected tumor cells as a virus-associated antigen in rats.

Renal cell cancer (RCC) cell lines, ACHN and KRC/Y, with or without exposure to interferons (IFNs), were examined for their susceptibility to lymphokine-activated killer (LAK) cells in relation to modulation of major histocompatibility complex (MHC) class I antigens on tumor cells. Flow cytometric analysis demonstrated constitutional expression of class I antigen on both cell lines, which was enhanced by IFN-alpha and -gamma, and was reduced by acid treatment at pH 3. A 4-h 51Cr-release cytotoxicity assay demonstrated that pretreatment of both cell lines with IFN-alpha and -gamma decreased their susceptibility to LAK cells. Although an inverse correlation between class I antigen expression and susceptibility to LAK cells has been reported by others, IFN and acid treatment demonstrated that the degree of class I antigen expression did not correlate with the susceptibility to LAK cells. These results suggest that clinically administered IFNs might induce protection of RCC to LAK cells, and that decrease of susceptibility might depend upon a mechanism different from the enhancement of class I antigens which is frequently expressed on RCC.

It is well known that Epidermal Growth Factor (EGF) is a cell-regulating factor for variety of tissues in vitro including normal and malignant cells. Furthermore, Takano et al reported that a decreased expression of EGF receptor in clones of human cancer KB cell line might be one of the pleiotropic properties of multidrug-resistant cells. However, both the influence of EGF on human urological cancer cell lines and the relation between EGF receptors and sensitivities of antitumor drugs on these cell lines have not been fully described. We have studied the effects of EGF on growth of 4 transitional carcinoma cell lines of bladder (TCCaB), 1 squamous cell carcinoma cell line of bladder (SCCaB), 5 renal cell carcinoma cell lines (RCC) and 3 prostatic carcinoma cell lines (CaP), as well as the relationship between the number of EGF receptors and drug sensitivities of these cell lines in vitro against methotrexate, vinblastine, adriamycin, cisplatin and etoposide (VP16). The present results determined by the in vitro colony forming efficiency method showed that exogenous addition of EGF to cell cultures at 0.1 ng/ml stimulated the growth of SCCaB by 169.0%, and at 1 ng/ml inhibited that of RCC by 2.9%-79.0%, relative to control. The more EGF receptors by 125I-EGF binding assay, the higher inhibition of VP16 on the growth of these cell lines. These results suggested that EGF stimulated the growth of SCCaB and inhibited the growth of RCC in vitro, and we found that these phenomena were correlated with neither the number of EGF receptors nor affinities of that receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The hypoxic cell fraction in tumors is considered to be responsible for radioresistance. Estimating the population of the hypoxic cell fraction in tumor could develop the effective means to predict radiosensitivity. In this study, nitroacridine (fluorescent dye) was tested to estimate hypoxic status in single cells and in spheroids. The oxygen concentration in the medium was measured by oxygen electrodes utilizing polarography method, mean while that in cells was calculated from the fluorescence intensity of the dye. The fluorescent spectra from cells showed the same pattern in spite of the changed oxygen concentration in medium, on the other hand its intensity was dependent upon the oxygen concentration. Using a fixed nitroacridine concentration and a fixed staining time, oxygen concentration of cells could be determined within range from 0.1 to 1.0% values. These values are almost the same as the oxygen concentration of the radioresistant tumor cells. Thus, the fluorescent method we used in this study is considered to be useful to estimate radioresistance of tumor. However, fluorescent intensity would alter when used different cell lines, because of different cellular activity of nitroreductase.

A study was made on 2 types of 131I-labeled anti-CA 19-9 and anti-CEA mouse monoclonal antibodies (IMACIS-1) against human cancer related antigen as to their usefulness in radioimmunoimaging. Tumor-bearing nude mice were used for comparison. The transplanted tumors (SW948, COLO 201) were clearly visualized 48-72 hours after administration of IMACIS-1. Tumor/blood ratio 72 hours after administration: 8.69 in COLO 201 and 5.70 in SW948, showing ca. 10-15 times as high as those in PC-3 and HEp-2. IMACIS-1 therefore is considered useful in radioimmunoimaging of cancer. Analysis was made by in vitro cell ELISA. As a result, both of the cells specifically reacted with anti-CA 19-9 but not anti-CEA.

In order to obtain killer lymphocytes that would have a strong cytotoxicity against autologous tumor cells, we conducted a cytotoxic assay of effector cells cultured from human peripheral blood lymphocytes (PBL) using 10 cultured lung cancer cell lines as target cells. We present this result. (1) Lymphocytes obtained by 17 days mixed lymphocyte tumor cell culture (MLTC: Fresh PBL from lung cancer patients was mixed with irradiated autologous tumor cells in a medium, and cultured for 3 days. Irradiated allogeneic lymphocytes were then added, and cultured for 14 days) were cytotoxic against 4 of the 10 autologous lung cancer cell lines. (2) Lymphocytes obtained by 14 days MLTC and 3 more days culture in a medium containing interleukin 2 were cytotoxic against all 10 of the autologous lung cancer cell lines. These lymphocytes proliferated to 4.13 times of the original cell number, and their surface marker was OKT3+. These killer lymphocytes are expected to be effective in adoptive immunotherapy as effector cells.

Flow cytometry (FCM) permits instantaneous determination of the percentages of cells in various phases of cell cycle using BrdU-PI double staining method, and allowing rapid evaluation of the effects of irradiation and anti-cancer drugs (ACNU, ADR, BLM) on the cell kinetics. In this study, the growth inhibition and changes in the cell kinetics after irradiation and chemotherapy were examined according to the growth curve analysis and BrdU-PI method to evaluate the usefulness of BrdU-PI method for assessment of the effect of the treatments. By the conventional method based on the DNA histogram, accurate determination of S cell fraction was difficult due to overlapping of the DNA contents of G1 cells and early S cells and those of late S cells and G2 cells. BrdU-PI double staining allowed direct differentiation of G1, S, and G2 + M cells, especially between G1-S and S-G2 + M cells. The analysis of cell kinetics using BrdU is advantageous in comparison to the conventional autoradiographic methods because it allows more rapid assay with very high sensitivity. By the present BrdU method, rapid transition to the G1-S phase was observed within 4 hours after exposure to radiation and anti-cancer drugs. This initial G1 arrest induced by irradiation was confirmed for the first time by the present BrdU-PI double staining. The present method is considered to be indispensable for evaluation of the percentage of S cells in the tumor tissue and analysis of cell kinetics after irradiation and chemotherapy against cancer.