Yoshiro Tanio

Association between angiotensin II receptor gene polymorphism and serum angiotensin converting enzyme (SACE) activity in patients with sarcoidosis

BACKGROUND Serum angiotensin converting enzyme (SACE) is considered to reflect disease activity in sarcoidosis. SACE activity is increased in many patients with active sarcoid lesions. The mechanism for the increased SACE activity in this disease has not been clarified. ACE insertion/deletion (I/D) gene polymorphism has been reported to have an association with SACE levels in sarcoidosis, but no evidence of an association between angiotensin II receptor gene polymorphism and SACE in this disease has been found. A study of the association of angiotensin II receptor gene polymorphisms with sarcoidosis was therefore undertaken. METHODS ACE (I/D), angiotensin II type 1 receptor (AGTR1), and angiotensin II type 2 receptor (AGTR2 ) gene polymorphisms were investigated by polymerase chain reaction (PCR) and SACE levels were measured in three groups of patients: those with sarcoidosis or tuberculosis and normal controls. RESULTS There was no difference in allele frequency of AGTR1 and AGTR2 polymorphism among the three groups. Neither AGTR1 nor AGTR2 polymorphisms were associated with sarcoidosis. SACE activity was higher in patients with sarcoidosis with the AGTR1 A/C genotype than in others. However, this tendency was not detected in patients with tuberculosis. CONCLUSIONS The AGTR1 allele C is associated with high activity of SACE in patients with sarcoidosis. It is another predisposing factor for high levels of SACE in patients with sarcoidosis and is considered to be an independent factor from the ACE D allele for high levels of SACE in sarcoidosis. This fact could be one of the explanations for the increased SACE activity in sarcoidosis.

Detailed deletion mapping of the short arm of chromosome 3 in small cell and non-small cell carcinoma of the lung

We constructed a detailed deletion map of the short arm of chromosome 3 (3p) for 55 lung cancer cases by using 17 restriction fragment length polymorphism (RFLP) probes. Initially, we examined 40 small cell lung cancer (SCLC) cases and found three regions of deletion at 3p25-26, 3p21.3 and 3p14-cen, suggesting the possibility of at least three different tumor-suppressor genes on 3p. In order to obtain more detailed deletion area, and to compare the pattern of 3p deletion, we also examined 15 non-small cell lung cancer (NSCLC) cases. Compared to NSCLC cases, most of SCLC cases have widespread deletion on 3p, suggesting multiple tumor-suppressor genes on 3p may be inactivated in this type of cancer. In 3p21.3 area, minimum overlapping area of deletion lays between two probes which are close to each other. These data will be useful to isolate the putative tumor-suppressor genes located on the chromosome 3p.

Adenovirus-mediated gene therapy specific for small cell lung cancer cells using a myc-max binding motif

Recent clinical trials of gene therapy for patients with thoracic cancers have shown that these treatments were well tolerated with minimal side effects and that we need to further enhance specificity as well as efficiency of gene transfer to target cancer cells. We previously reported that myc‐overexpressing SCLC cell lines became selectively sensitive to ganciclovir (GCV) by transducing the herpes simplex virus thymidine kinase (HSV‐TK) gene under the control of the Myc‐Max response elements (a core nucleotide sequence, CACGTG) and that this construct (MycTK) could be utilized to develop a novel treatment against chemo‐radio‐resistant SCLC. We report here in vivo antitumor effects and safety of a replication‐deficient adenoviral vector containing the Myc‐Max binding motif (AdMycTK) on SCLC cells. In vitro infection with AdMycTK selectively rendered myc‐overexpressing SCLC cell lines 63‐ to 307‐fold more sensitive to GCV. In vivo injections with AdMycTK followed by GCV administration markedly suppressed the growth of myc‐overexpressing tumors established in the subcutis or in the peritoneal cavity of athymic mice. On the other hand, infection with AdMycTK did not significantly affect either in vitro GCV sensitivity of the cells expressing very low levels of the myc genes or the growth of their subcutaneous tumors. Moreover, we observed no apparent side effects of this treatment including body weight loss or biochemical abnormalities in contrast to the treatment with AdCATK that conferred strong but nonspecific expression of the HSV‐TK gene. These results suggested that AdMycTK/GCV therapy is effective on SCLC patients whose tumors overexpress myc family oncogenes.

Complementary DNA sequence encoding the major neural cell adhesion molecule isoform in a human small cell lung cancer cell line

The neural cell adhesion molecule (N-CAM), a member of the immunoglobulin gene super-family mediating homophilic cell-cell adhesion in a neuroendocrine system, is preferentially expressed in human small cell lung cancer (SCLC). Immunoprecipitation of a panel of SCLC cell lines by monoclonal antibodies (mAbs) specific for N-CAM detects mainly the 145-kDa isoform. This result was correlated with Northern blotting where a single 6.2-kb mRNA was detected in nine SCLC cell lines. To determine cDNA sequence encoding the N-CAM isoform, we selected several cDNA clones encoding N-CAM isolated from OS2-R, a SCLC cell line established in our laboratory. Based on the analysis of the full-length cDNA obtained from two clones, the sequence of this 145-kDa isoform was shown to be essentially identical to that of the 140-kDa N-CAM isoform of neuroblastoma except for a single base pair changed at position 1620 without changing amino acid encoded.

A Phase II Study Directed by a Clinical Pathway for Carboplatin and Weekly Paclitaxel in Previously Untreated Patients with Unresectable Non-Small Cell Lung Cancer

The objective of this phase II study was to evaluate the efficacy and safety of carboplatin and weekly paclitaxel in previously untreated patients with unresectable non-small cell lung cancer. In addition, the clinical pathway intensified the management of chemotherapy including the assessment of efficacy, safety and implementation of treatment and patient education. Patients received paclitaxel at a dose of 70 mg/m2 on days 1, 8 and 15 and carboplatin (area under the curve of 6) on day 1 and every 28th day thereafter. Fifty-eight patients were enrolled. A median of 3 cycles (range 1–6) were administered. Twenty-eight cases showed objective responses (48.2%), including 2 complete (3.4%) and 26 partial responses (44.8%; 95% confidence interval 35.4–61.1). The median survival time was 663 days, and the 1-year survival rate was 59.9%. Nineteen patients (32.8%) had grade 3, and 4 patients (6.9%) had grade 4 neutropenia. Nine patients (15.5%) experienced ≧3 grade nonhematological toxicities. There were no treatment-related deaths due to this study. Carboplatin and weekly paclitaxel combination chemotherapy might be an alternative treatment selection in patients with unresectable non-small cell lung cancer.

Chemo‐radioresistance of Small Cell Lung Cancer Cell Lines Derived from Untreated Primary Tumors Obtained by Diagnostic Bronchofiberscopy

New cell lines of small cell lung cancer (SCLC) were established from specimens of untreated primary tumors biopsied by diagnostic bronchofiberscopy. The advantage of this method was ease of obtaining specimens from lung tumors. Establishment of cell lines was successful with 4 of 13 specimens (30%). Clinical responses of the tumors showed considerable variation, but were well correlated with the in vitro sensitivity of the respective cell lines to chemotherapeutic drugs and irradiation. One of the cell lines was resistant to all drugs tested and irradiation, while another was sensitive to all of them. Although the acquired resistance of SCLC is the biggest problem in treatment, the natural resistance to therapy is another significant problem. Either acquired or natural, resistance mechanisms of SCLC may be elucidated by the use of such cell lines derived from untreated tumors. This method and these SCLC cell lines are expected to be useful for the serial study of biologic and genetic changes of untreated and pre‐treated tumors, or primary and secondary tumors.

High Sensitivity to Peripheral Blood Lymphocytes and Low HLA‐class I Antigen Expression of Small Cell Lung Cancer Cell Lines with Diverse Chemo‐radiosensitivity

Three cell lines of small cell lung cancer (SCLC), which were established from specimens of untreated primary tumors biopsied by diagnostic bronchofiberscopy, were analyzed for immunological characteristics. These cell lines showed considerable heterogeneity in chemo‐radiosensitivity, which was well correlated with clinical responses of the respective tumors, but their HLA‐class I antigen expressions were equally depressed and they were susceptible to peripheral blood lymphocytes (PBL) and lymphokine‐activated killer (LAK) cells, irrespective of their diverse chemo‐radiosensitivity. Treatment of the cell lines with recombinant immune interferon (rIFN‐γ) increased their HLA‐class I antigen expression and conversely depressed PBL sensitivity but not LAK sensitivity. This inverse relationship between HLA‐class I expression and PBL susceptibility was also demonstrated using other pairs of autologous PBL and SCLC cell lines. rIFN‐γ changed neither HLA‐class H antigen nor SCLC‐spccilic antigen expression under the same experimental conditions. In vitro immunization of allogeneic peripheral blood lymphocytes with rIFN‐γ ‐treated SCLC cells induced allo‐specific killer cells which lysed rIFN‐7‐treated more strongly than non‐treated SCLC cells. These results suggest that reduced HLA‐class I antigen expression of SCLC could protect the cancer from attack of killer T cells in spite of the higher sensitivity to PBL or LAK cells.

Two monoclonal antibodies against small-cell lung cancer show existence of synergism in binding

SummaryMurine IgG1 monoclonal antibodies (mAbs), ITK-2 and ITK-3, were generated against a small-cell lung cancer (SCLC) cell line. Enzyme-linked immunosorbent assay using a variety of established cell lines as substrates, immunoperoxidase staining of freshly frozen tissue sections, and fluorescence-activated cell sorter analysis of peripheral blood leukocytes showed that these mAbs recognize a part of the SCLC-associated cluster 1 antigen. In immunoprecipitation studies, both ITK-2 and ITK-3 bound to a 145-kDa glycoprotein of SCLC cell membrane extracts, as did MOC-1 and NKH-1, which both recognize the cluster 1 antigen. However, because the binding of125I-labeled ITK-2 to SCLC cells was not inhibited by MOC-1 or NKH-1, the binding site of ITK-2 on SCLC cells appeared to be different from that of either MOC-1 or NKH-1. Unexpectedly, binding of125I-labeled ITK-2 to SCLC cells increased in the presence of ITK-3. This ITK-3-induced increase in ITK-2 binding was due partly to an increase in the number of binding sites for ITK-2 on SCLC cells. Addition of ITK-3 may, therefore, improve the effectiveness of ITK-2-based tumor detection or therapy.

Characterization and Purification of an Immunosuppressive Factor Produced by a Small Cell Lung Cancer Cell Line

The present study was undertaken to determine whether small cell lung cancer (SCLC) cell lines produce immunosuppressive factors and, if they do, to characterize the factors. The supernatants of SCLG cell lines, H69 and N857, inhibited not only the blastogenic response of human peripheral blood lymphocytes (PBL) to phytohemagglutinin or concanavalin A, but also the cytotoxic activity of lymphokine‐activated killer cells. Neither was inhibited by supernatants from non‐SCLC cell lines PC9, QG56, and A549. The immunosuppressive activity of H69 supernatant was stable upon heating to 56°C for 60 min, but labile when heated to 70°C for 10 min. The activity was abolished after dialysis at pH 2.0 or pH 11.0, but not at pH 4.5 or pH 9.0. Digestion with trypsin or proteinase eliminated the immunosuppressive activity, whereas treatment with neuraminidase, mixed glycosidase, DNase or RNase had no effect, suggesting that the immunosuppressive activity in H69 supernatant is due to a protein factor. This H69‐derived immunosuppressive factor was isolated by ion exchange chromatography using a gradient of 0.04 to 0.08 M NaCl solution. Gel filtration and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis showed the factor to have molecular weights of 98 kD and 102 kD, respectively. These results suggest that SCLC cells produce a potent immunosuppressive factor which may account for the immune deficiency in SCLC patients.

Augmentation of Murine Lymphokine-activated Killer Cell Induction by a Factor Produced by Nocardia rubra Cell Wall Skeleton-stimulated T Cells

Four‐hour exposure of C3H/HeN mouse spleen cells to Nocardia rubra cell wall skeleton (N‐CWS) before 4‐day culture with a suboptimal dose of human recombinant interleukin 2 (rIL 2) augmented the induction of lymphokine‐activated killer (LAK) cell activity, whereas the treatment with N‐CWS alone induced no cytotoxicity. In accordance with this, the IL 2 binding activity of spleen cells was augmented by combined stimulation with N‐CWS and rIL 2. The augmented cytotoxicity was mediated by Thy‐1.2+, Lyt‐1.1−, Lyt‐2.1− and asialo GM1+ cells. Cell cultures in diffusion chambers revealed that N‐CWS‐treated spleen cells produced a LAK cell induction‐helper factor (LAK‐helper factor, LHF) when cultured with rIL 2. The LHF production required Thy‐1.2+, Lyt‐1.1+, Lyt‐2.1+ and asialo GM1− cells, and the coexistence of unstimulated accessory cells was also essential for the LHF production. Cells responding to both LHF and rIL 2 to generate LAK activity were Thy‐1.2−, Lyt‐1.1−, Lyt‐2.1− and asialo GM1+. The culture fluid of spleen cells stimulated with both N‐CWS and rIL 2 contained no tumor necrosis factor (TNF) activity, and the additional stimulation with N‐CWS caused no production of either IL 2 or interferon (IFN). Murine recombinant interleukin la (Mu‐rIL 1α) could not replace the augmentative effect of N‐CWS on LAK cell induction. These results suggest that in the presence of rIL 2, N‐CWS stimulates murine T cells to produce LHF that is probably distinct from IL 1, IL 2, TNF and IFN.