Recently, a new therapeutic approach to leukemia has been developed by induction of differentiation of leukemic cells. To improve this therapy, various factors involved in in vivo induction of differentiation of leukemia cells should be clarified. Clinical studies showed that inducers of differentiation of leukemia cells including retinoic acid, hexamethylene bisacetamide and some chemotherapeutic drugs induced remission in patients with acute myelogenous leukemia. Clonal analysis of the matured granulocytes revealed that the effectiveness of these drugs was due to their ability to induce differentiation of the leukemic cells rather than their cytocidal activity. On the other hand, various types of agents have been found to induce in vitro differentiation of leukemia line cells and fresh human leukemic cells. To evaluate therapeutic efficacy of these differentiation-inducing agents, pre-clinical experiments using animal model and tests for in vivo inducibility of differentiation of the leukemia cells are essential. We are developing an experimental system to monitor in vivo induction of differentiation of murine leukemia cells inoculated into syngeneic mice.

Twenty-one human ovarian epithelial carcinomas (13 serous, 3 mucinous, 2 endometrioid and 3 clear cell type) were examined for allelic losses of p53 tumor suppressor gene, pYNZ22 or pTHH59 on chromosome 17. High-molecular-weight DNA was extracted from ovarian tumor tissues and normal ones obtained from the same patients, and was subjected to Southern blot analysis. The filters were hybridized with p53 cDNA probe or two VNTR probes on chromosome 17(pYNZ22, pTHH59). By comparing the hybridized band pattern of the normal tissue with that of the tumor, loss of heterozygosity (LOH) was detected. Frequent LOHs were detected in 5 of 14 informative cases (36%) on p53 and in 9 of 20(45%) on pYNZ22, but LOH on pTHH59 was observed less frequently than those of p53 and pYNZ22 (one of 7 cases: 14%). These results suggest that allelic loss of p53 may play an important role in the development and/or progression of ovarian carcinomas.

Expression of the human papillomavirus (HPV) gene was examined in HPV-positive laryngeal tumors. Moreover, the activity of the HPV long control region (LCR) was tested in cultured laryngeal epithelial cells. HPV-11 early genes were heterogeneously expressed in adult laryngeal papillomas. We found one laryngeal carcinoma case in whom the HPV-16 transforming genes, E6 and E7, were expressed. Both HPV-11 and -16 LCRs were active in cultured laryngeal epithelial cells from vocal cords. These results suggest that laryngeal epithelial and tumor cells are target cells for HPV gene expression.

Using reverse transcription polymerase chain reaction, we determined mRNA expression of topoisomerase (topo) II alpha and beta in adriamycin- and etoposide-resistant small cell lung cancer sublines, SBC-3/ADM 100 and SBC-3/ETP. The expression of topo II alpha mRNA decreased substantially in SBC-3/ADM 100 and SBC-3/ETP as compared with the parent cell line, SBC-3; 0.71-fold in the former and 0.38-fold in the latter. Similarly, that of topo II beta mRNA decreased to an extent of 0.68-fold in SBC-3/ADM 100 and 0.28-fold in SBC-3/ETP as compared with the parent cell line. SBC-3/ADM 100 and SBC-3/ETP were highly resistant to topo II inhibitors such as daunorubicin, epirubicin, pirarubicin, mitoxantrone, and teniposide. However, SBC-3/ADM 100 showed a less resistance to aclarubicin, and SBC-3/ETP was as sensitive to the drug as was in the parent cell line. The resistance to topo II inhibitors excluding for aclarubicin might be partially explained by the decreased expression of topo II alpha and beta mRNA.

Matrixmetalloproteinases (MMP), such as type IV collagenases and interstitial collagenases, play an important role in tumor invasion and metastasis. And tissue inhibitor of metalloproteinases (TIMP) inhibit collagenolytic activity of these enzymes. We investigated the gene expressions of MMP-9 (92 kDa type IV collagenase), MMP-2 (72 kDa type IV collagenase), TIMP-1 and TIMP-2 in bladder cancers by Northern blot and slot blot hybridization. The mRNA levels of MMP-2, TIMP-1 and TIMP-2 increased in the cases with invasion and metastasis of bladder cancers. These findings suggest that MMP-2 acts as a regulator of the invasion and metastasis of bladder cancers. The MMP-2/TIMP-2 ratio increased as tumor invasion and metastasis progressed, suggesting that an imbalance in the MMP and TIMP ratio promote the invasion and metastasis of bladder cancers. And we also investigated the gene expressions of c-fos that activate the collagenase genes, and there was a correlation between c-fos and MMP-2 in gene expressions. It is suggested that fos gene may play an important role for the invasion and metastasis in bladder cancers.

Philadelphia chromosome (Ph1) is detected in more than 95% of chronic myelogenous leukemia (CML) and approximately 20% of adult acute lymphocytic leukemia (ALL). In order to discriminate Ph1-positive ALL from Ph1-positive CML as a clinical entity, I studied on biological and genetic characteristics of Ph1-positive ALL cells. Two cases out of 11 Ph1-positive ALL showed hybrid leukemia phenotypes; in one hybrid case simultaneous proliferation of lymphoid and myeloid blast cells was observed and contained rearranged alleles of heavy chain genes, thus indicating that both blast cells might originate from a common precursor. Two Ph1-positive ALL cell lines (TOM-1 and ALL-MIK) were established from two patients and were investigated for their differentiation potential in vitro. Both cell lines showed the potency to differentiate into monocytic lineage cells, thus suggesting that these Ph1-positive ALL cells might reside at the stage of multipotent progenitor cell along hematopoietic cell differentiation. As to Ph1-chromosome, 4 out of 9 Ph1-positive ALL cases showed rearrangements within the classical sequence (M-bcr), similar to those in CML cases. Two out of 5 cases without rearrangement of M-bcr showed breakpoints in the first intron of the BCR gene. In the rest of 3 cases, BCR-ABL rearrangement was not detected by Southern analysis. However, a leukemic cell line established from one of these patients (TOM-1) were contained P190bcr-abl mRNA as analyzed through RT-PCR. Thus, breakpoints of the BCR gene in Ph1-positive ALL cases were heterogenous, in contrast to those of CML. Then, I investigated whether or not the activation of transforming genes other than BCR-ABL might be involved in pathogenesis of Ph1-positive ALL. Three out of 15 Ph1-negative ALL cases showed the mutations of RAS gene by the PCR. However, no activated oncogene was detected in Ph1-positive ALL cases by both DNA transfection assay and PCR.

The retinoblastoma susceptibility gene (RB) and p53 gene are now known to be the prototypes for a class of tumor suppressor genes. Both genes act as a regulator of cell cycle transition at G1/S in many types of cell lineages. Underphosphorylated form of RB protein (Rd) acts as a growth suppressor by blocking exit from G1 through a specific binding to E2F or promoter region of certain growth-associated genes. Phosphorylation of Rb can be viewed as inactivating Rb and allowing cell cycle progression to occur. Differentiation of hematopoietic cell is accompanied with the loss of ability to phosphorylate Rb, indicating that Rb plays an important role in hematopoietic cell growth and differentiation. Abnormalities of RB gene may, therefore, predispose to the development of hematologic malignancies. DNA rearrangement was reported to be present in 1.5-12.1% of cases with primary leukemias, and the absence of RB protein was also observed in 6.3-23.2%. The abnormalities of p53 gene were also frequently observed in hematologic malignancies. DNA rearrangement of p53 was observed in 20-30% of the cases with blastic crisis of CML. Point mutation at the "hot spot" was reported in many types of leukemias, especially in cell lines established from these cases.

Current progressive chemotherapy and transplantation induce not only remission but complete cure. By this reason, the detecting system for MRD must be established in complete remission phase. In this study, we showed the detecting for MRD by molecular biology techniques in the patients with non-Hodgkin's lymphoma, CML, B-ALL and AML. 1) Malignant cells could be detected in peripheral blood or bone marrow cells by Southern blot analysis in 15 of 30 patients with non-Hodgkin's lymphoma. 2) In CML patients, BCR/ABL fusion gene was positive by RT-PCR for 6 months after BMT, 4 patients became undetectable for 7 months after BMT. 3) Rearrangement of Ig H has been disappeared in 12 months after achieved complete remission the patients with B-ALL. In conclusion, the detection for MRD remain an important goal for therapy including chemotherapy and bone marrow transplantation in hematopoetic malignancy.

A lot of alterations of oncogenes have been reported to occur in human cancers in vivo. The types of alterations which occur most frequently are point mutations of ras genes, amplifications of myc and erbB gene families and rearrangement of oncogenes by chromosomal translocation. Each type of gene alteration discloses the specificity not only to organ and cell types from which cancer originated but also to etiological background and clinical aggressiveness of cancer. These gene alterations are shown to activate oncogenes and suggested to be involved in the mechanism of genesis and/or progression of human cancers.

Of 140 cases of mediastinal neoplasms in our hospital, histological diagnosis was confirmed in 129 cases. We examined the methods of preoperative biopsy with those 129 cases. Biopsy had been performed in 25 cases. Mediastinoscopy was performed in seven cases, needle biopsy in eight cases, lymph node biopsy in eight cases, esophageal biopsy using a gastrofiberscope in one case, transbronchial biopsy using a bronchoscope in one case. The true positive rates of those methods were 100% for both mediastinoscopy and lymph node biopsy, and 75% for needle biopsy. Preoperative misdiagnosis occurred in two cases of needle biopsy. The postoperative histological diagnosis was malignant lymphoma in both cases. We performed gene analysis of the immunoglobulin heavy chain gene, light chain kappa and lambda genes, and the T-cell receptor beta gene by use of biopsied specimens, and we found rearrangement bands of these genes in the cases of malignant lymphoma. Therefore, we summarize that gene analysis is a reliable method if malignant lymphoma is suspected. If a needle biopsy is performed under CT guidance, the needle is sure to puncture the tumor. We concluded, therefore, that if a tumor is located in the anterior mediastinum, CT-guided needle biopsy should be performed first of all. Mediastinoscopy is a useful method if the tumor is located in the mid-mediastinum.

The authors report a de novo AML (M2) patient associated with 5q- as the sole karyotypic abnormality. A 76-year-old woman was referred to our hospital because of anemia and leukocytosis. On examination a neck lymph node was enlarged, but neither the liver nor the spleen could be palpated. The hemoglobin level was 7.1g/dl, the mean corpuscular volume 102fl and the white-cell count was 256.1 x 10(3)/microliters with 87% blast cells. The platelet count was 10.9 x 10(4)/microliters. The bone marrow was hypercellular with 79.8% blast cells and showed dysmegakaryocytopoietic features (hypolobulation, multiple separated nuclei and micromegakaryocytes). Blast cells gave a positive reaction for peroxidase and alpha NB esterase which was not blocked by NaF. The diagnosis of AML (M2) was made but she died before chemotherapy. Autopsy revealed general hemorrhagic tendency and leukemic cell infiltration. Chromosome analysis of the bone marrow showed 46,XX,del(5) (q13q31). Electron micrographs revealed increase of micromegakaryocytes as small as myelocytes and aggregation of demarcation membranes in some megakaryocytes. This may suggest that some molecular changes, instead of karyotypic evolution, contributed to a leukemic transition from the 5q- syndrome to AML with 5q- as the sole abnormality.

We established four renal cell carcinoma (RCC) cell lines (HANKS), namely the primary tumor (HANKS-Pr), metastasized to the lung (HANKS-Lu), liver (HANKS-Li) and lymph node (HANKS-LN) derived from a patient with advanced RCC, and analyzed their characters. Each had an epithelial morphology and exhibited multilayering. These cell lines have been maintained for more than 36 months and over 100 in vitro passages. In karyotype analysis, the common aberration in the four cell lines was marker chromosome t (3;18) (p13;q21). In soft agar culture, HANKS-Pr showed the lowest growth. Furthermore, we found high level expression of major histocompatibility complex (MHC) class I antigen on HANKS-Pr and HANKS-Lu, and low expression of MHC class II antigen on four cell lines. HANKS-LN had transplantability in nude mice. We determined the different biological properties among HANKS cell lines stemming from the same origin.

The point mutation of K-ras gene at codon 12 was investigated in 11 cases of gall bladder carcinoma, 10 cases of extrahepatic bile duct carcinoma, 2 cases of intrahepatic bile duct carcinoma, and 4 cases of ampullary carcinoma by modified two-step polymerase chain reaction which employed paraffin embedded materials. The results revealed that there were point mutations in 6/11, 10/10, 2/2, and 4/4, respectively. Judging from the ratio of density of wild and mutant band, not all cancer cells in the tissue section contained the mutation. So it was suggested that the normal cells were initiated for the malignant transformation by ras gene mutations and then, selection might occur against cells containing ras mutations during progression of the tumor. Modified two-step polymerase chain reaction was markedly useful for detecting mutation even at low frequency among tumor cells.

Eleven tongue papillomas and 25 tongue carcinomas were produced in rats by the oral administration of 0.001% 4-nitroquinoline 1-oxide (4 NQO) in drinking water, and tumor kinetics were investigated by both DNA cytofluorometry and Ag-nucleolar organizer regions (Ag-NORs) staining. The histopathologic characteristics, the mean Ag-NOR number per cell and DNA ploidy patterns were compared. The mean Ag-NOR number was least in normal epithelium, more numerous in papillomas and highest in squamous cell carcinomas. The differences were statistically significant. All cases of normal epithelium and papilloma showed a diploid pattern. Eighteen cases of the squamous cell carcinomas (72%) showed a diploid pattern, and 7 cases (28%) exhibited a diploid plus tetraploid pattern. There was a significant difference in the mean Ag-NOR number between the two groups of squamous cell carcinomas. These results suggest that the mean Ag-NOR number may reflect the cell kinetics in the process of carcinogenesis and polyploidization of carcinomas.

As predictors of tumor response by radiotherapy, tumor with or without c-myc amplification and DNA histogram pattern were analyzed and evaluated for patients' prognosis. There was no relation between c-myc amplification and DNA ploidy pattern in this study. These factors also showed no relation to response rate and survival. But all of the tumors with c-myc amplification and aneuploid pattern exhibited a complete response by radiotherapy. The tumors with c-myc amplification and aneuploid pattern may be more radiosensitive than without amplification and diploid. These data suggested that c-myc amplification and ploidy pattern might be a predictive parameter before radiotherapy.

The authors previously reported the advantages of a collagen gel embedded culture system for chemosensitivity tests for cancer. In this report, the chemosensitivities of surgically resected specimens were evaluated by the collagen gel embedded culture system and compared with the DNA ploidy pattern, measured by flow cytometry. The chemosensitivity and DNA ploidy pattern were determined in 11 patients with lung cancer, 8 with gastric cancer and 46 with colorectal cancer. Anticancer agents were MMC and CDDP at Cmax for one hour of exposure, and 5-FU, VDS, VP-16 and ADM at one tenth the Cmax for 24 hours of exposure. Results were compared with those of DNA histogram. In eight lung cancers which were demonstrated to be sensitive by the collagen gel system, 5 showed DNA aneuploidy (DA) and 3 DNA diploidy (DD). Seven cases (87.5%) of gastric cancer were demonstrated to be sensitive with the collagen gel system. Two of them showed DA and five DD. On the other hand, 19 cases (41.3%) of colorectal cancer were found to be sensitive, and 7 of them showed DA and twelve DD. Lung cancer and gastric cancer exhibiting aneuploidy demonstrated sensitivity with the collagen gel system, but the rate of sensitivity was only 28% in colorectal cancer, and even aneuploidy cases showed a low sensitivity.