NORs (Nucleolar Organizer Regions) are loops of DNA encoding ribosomal RNA, which reflect cellular activity. The Ag-NORs staining was applied to paraffin sections of 10 normal pancreas, 10 chronic pancreatitis and 23 pancreatic cancer, with 17 gastric and 18 colonic cancers. We calculated the mean Ag-NORs number and Ag-NORs index, and defined T/N ratio. The mean Ag-NORs number was compared with the data obtained by Flow cytometry (FCM) in pancreatic carcinoma. A significant difference in Ag-NORs index was found between benign (normal: 1.86 +/- 0.38, pancreatitis: 2.14 +/- 0.53) and malignant tissues (4.02 +/- 1.19) of the pancreas (p < 0.01). Compared with the data by FCM, there was a significant correlation between the mean Ag-NORs number and the percentage of S-phase cells in pancreatic carcinoma (r = 0.59, p < 0.01). Ag-NORs index in pancreatic carcinoma showed significantly lower value than that in cancer of stomach (6.35 +/- 1.32) and colon (7.66 +/- 1.35), but there was no difference in T/N ratio among them. In conclusion, the Ag-NORs staining is useful for analyzing proliferating activity in pancreatic carcinoma. The proliferating potential in pancreatic carcinoma is lower than that in gastric and colonic ones. This may be due to the low proliferating potential of the background mucosa from which pancreatic carcinoma will arise.

Forty fresh gastric cancer specimens were examined by immunohistological staining using anti-BrdU monoclonal antibody, and simultaneously the specimens were studied by DNA ploidy pattern, EGF with flowcytometer and the immunohistological staining. For EGF study, the same 40 gastric cancer formalin fixed specimens were used. The results of flowcytometric measurement were divided into diploid and aneuploid patterns. BrdU positive stained cancer cells were observed in growing border area than in the center of tumor, and histologically in P(+), n(+), ps(+), INF gamma, and in scirrhous type, deep spread type and advanced stage. This data suggested that the BrdU labeling index seemed to be related to invasion, proliferation, and metastasis of cancer cells. However the positive rates of EGF were higher in ps(+), deep spread type and BrdU positive type but not in P(+), n(+), and EGF was considered to related to invasion but not to be related to metastasis. Although aneuploid pattern cancer showed high BrdU labeling index in ps(+), diploid pattern didn't indicate such tendency. High BrdU positive rate aneuploid cancer were seemed to grow quickly, advance in short period and own worse character. Further investigation would be necessary.

Expression of the TS and DNA polymerase beta genes as a drug resistant-gene were compared with the human colon carcinoma cell line HCT8S and a subline that was 4.5 fold resistant to cisplatin. Resistant cells (HCT8DDP) exhibited 3.4 fold and 2.5 fold increase in mRNA for both TS and DNA polymerase beta gene when compared with the parent cells by Northern blotting analysis, respectively. The RT (reverse transcription)-PCR (polymerase chain reaction) method has been modified to quantify a sequence of a drug resistance gene. This method exhibited greater sensitivity than conventional methods (Northern blotting analysis), requiring less than 1 fetogram of mRNA from cell lines. The RT-PCR assay could be an effective device in the early detection of resistance to chemotherapy.

Various functioning and non-functioning tumors arise from endocrine glands in both the sporadic and familial forms and pathophysiology of the tumors is variable due to differences in the sort of tumor-bearing endocrine organs and in the amount of hormones released. In this paper, gene abnormalities in growth hormone (GH)-secreting pituitary adenoma, ectopic GHRH-producing tumor, multiple endocrine neoplasia (MEN) and ectopic parathyroid hormone (PTH)-producing tumor are documented in relation to etiology and pathophysiology. GH-secreting pituitary adenoma is heterogeneous in clinical features, pathological findings and GH responses to various secretagogues. A point mutation of codon 201 of Gs alpha gene was observed in 2 out of 45 GH-secreting pituitary adenomas (4.4%), but no point mutation of Gi2 alpha gene was found. Pituitary tumors may occur at any stage of differentiation from the totipotent cells to mature anterior pituitary cells, and the mutations of Gs alpha and H-ras genes as well as loss of heterozygosity (LOH) found on chromosome 11 in some adenomas must be involved in their tumorigeneses. Since 1959, 34 patients with ectopic GHRH-producing tumor associated with acromegaly have been reported. In our case of MEN type 1, the paradoxical rise of plasma GH after TRH or glucose administration disappeared after resection of the tumor. The tumor cells showed neither rearrangement nor amplification of GHRH gene and 20 oncogenes including ras, myc, and erb. Only LOHs of HRAS1 and D11S151 were detected in this tumor, but no point mutation was found in HRAS1 gene. Therefore, a kind of tumor suppressor gene may be involved in the tumorigenesis of the tumor in addition to inactivation of MEN-1 locus. In MEN-1 patients, we reported LOH on chromosomes 1, 9, 11 and 16, while we reported point mutation as being present only in Gs alpha gene on chromosome 20. This point mutation was found specifically in GH-secreting pituitary adenoma but not in hyperplastic parathyroid and pancreas adenoma. These data suggest that in MEN-1 patients tumorigenesis occurs and advances from hyperplasia and adenoma to cancer during multistep changes of genes such as inactivation of MEN-1 gene and other tumor suppressor genes and activation of oncogenes. Ectopic PTH-producing tumor was first reported by us in 1989, and this was followed by 2 papers. These patients showed a disturbance of consciousness and high levels of serum calcium and plasma PTH.(ABSTRACT TRUNCATED AT 400 WORDS)

Several rodent studies based on molecular biology have suggested that accumulation of genetic alterations in cancer-associated genes is required to convert a normal cell into a malignant cell. Activation of oncogenes and inactivation of tumor suppressor genes appear to be involved in carcinogenesis. In renal cell carcinomas, we have recently implied that the presence of tumor suppressor genes at chromosome 3p13-14.3 and 21.3, the regions where are also commonly deleted in adenocarcinoma of the lung; at chromosome 5q21, the region where the MCC (mutated in colorectal cancer) gene and APC (adenomatous polyposis coli) gene are located; at chromosome 6q27; and at 10q 21-23. We have also indicated that genes on 3p is probably important for development of RCCs and genes on 5q, 6q, and 10q may be associated with progression of RCCs.

A case of malignant histiocytosis with rearrangements of both T-cell receptor and immunoglobulin genes. The patient was a 69 year-old woman suffering from high fever, which was unresponsive to the administration of various antibiotics and steroids for more than two weeks. Laboratory findings on admission revealed disseminated intravascular coagulopathy and liver dysfunction. The bone marrow examination showed an increased number of giant cells. Some of the giant cells had phagocytosis of various blood cells and were cytochemically stained with non-specific esterase, but not with myeloperoxidase and PAS. Immunohistochemical study revealed that alpha 1-antitrypsin alpha 1-antichymotrypsin, lysozyme and CD15 were all detected in the cytoplasm of some giant cells while CD30 was not detected. Of interest was the rearrangements of the T-cell receptor, Ig heavy chain and kappa chain genes on bone marrow mononuclear cells demonstrated by Southern blot analysis.

A 73-year-old man was admitted with severe abdominal fullness. Physical examination suggested much ascites. Laboratory data revealed that BUN was 39 mg/dl, Cr was 2.2 mg/dl, UA was 19.7 mg/dl, and LDH was 1,569 U/l. In ascites, there were many cells with large nuclei and many vacuoles. Immunocytological characterization indicated that most of these cells were B lineage. And diagnosis of malignant lymphoma, the patient was treated with VEPA therapy. However, he soon died with acute renal failure. We examined the possibility of gene rearrangements between the c-myc and immunoglobulin genes and found that c-myc gene was rearranged at the first intron, and joined with immunoglobulin JH locus as head to head. These results suggested that the patient carried Burkitt's lymphoma of the American type.

The chromosome der(1;7) (q10;p10) is a derivative chromosome consisting of the short arm of chromosome 7 and the long arm of chromosome 1. We observed this abnormality in three patients with acute myeloblastic leukemia (AML), myelodysplastic syndrome (MDS), or myeloproliferative disorder (MPD). Case 1 was a 76-yr-old male with a history of IgG myeloma treated with melphalan, cyclophosphamide, vincristine, and prednisolone (MEVP). AML-M1 developed one and half years after discontinuation of the MEVP therapy. Case 2 was a 39-yr-old male with MDS. Case 3 was a 56-yr-old male with refractory anemia with excess of blasts in transformation that evolved from primary myelofibrosis. Chromosome analyses revealed der(1;7) (q10;p10) in bone marrow cells of the three patients. All patients failed to respond to chemotherapy, and died within four months after the diagnosis. Thus, der (1;7) (q10;p10) may indicate a very poor prognostic outcome in patients with malignant hematologic disorders.

Metastasis is the most critical problem for the oncologists to treat cancer patients. The processes of cancer invasion and metastasis consist of a long series of sequential, interrelated, and complicated steps. Understanding the mechanisms responsible for cancer invasion and metastasis is, therefore, a primary goal of cancer research and will produce new approaches to control and inhibit cancer metastasis. Massive investigations are being carried out for cancer metastasis. Recently many investigations on the research fields other than cancer result in being related with metastasis research. We reviewed the current findings, including our data, on the research for cancer metastasis and discussed the current status of the research.

We analyzed the alteration of int-2, c-erbB-2 and EGFR genes in 32 cases of transitional cell carcinoma of the urinary tract, 15 cases of renal cell carcinoma and 14 cases of prostatic carcinoma by Southern blot hybridization method. Three- to 12 fold amplification of int-2 gene was observed in 4 (12.5%) of 32 transitional cell carcinomas. Of these 4 cases 3 were G3 tumor with muscle invasion and the remaining was G1, pTa tumor with subsequent recurrence of multiple tumors. The other 2 cases (6.3%) with invasive transitional cell carcinoma showed amplification of c-erbB-2 gene. Neither amplification nor gross rearrangement of EGFR gene was detected in transitional cell carcinoma. On the other hand, renal cell carcinomas and prostatic carcinomas had neither amplification nor gross rearrangement of these 3 genes. These results suggest that the int-2 gene located in chromosome locus 11q13 and the c-erbB-2 gene have a specific role in carcinogenesis and in progression of transitional cell carcinoma through their gene amplifications.

The 5-year survival of lung cancer patients is about 30% in Japan. One of the reasons for the poor prognosis seems to be drug resistance. It has been reported that certain types of oncogenes, such as ras, myc and fos, may play an important role in drug resistance. The myc protein forms a sequence-specific DNA-binding complex with Max and may act as a transcription factor; thus, it may be possible that myc family oncogenes are involved in DNA synthesis and repair processes mediating drug resistance. We report here that L-myc oncogene may be involved in the transition from drug-sensitive to drug-resistant phenotype of a certain small cell lung cancer cell line.

Three large cell carcinoma cell lines were established from tumors of lung cancer patients. The cell lines were named NUTLC-2, NUTLC-4 and NUTLC-5 and they were found to have the following biological characterization. 1) By chromosomal analysis, the tumor cells of two of the cell lines (NUTLC-2 and NUTLC-5) were human-origin cells. Numbers of chromosomes of these cells ranged from 52 to 59 in NUTLC-2 and from 68 to 75 in NUTLC-5, with the modal numbers of 56 and 71, respectively. 2) Primary tumor resected from the patient with lung cancer was heterotransplanted into the subcutis of a nude mouse. NUTLC-4 cell line was established in vitro from the tumor in nude mouse and the tumor cells were found to be mouse-origin cells by chromosomal analysis. Human DNA was not detected by Alu analysis. 3) The tumor cells of three cell lines could be heterotransplanted subcutaneously into nude mice. However, no natural distant metastasis in nude mouse was observed. 4) Drug sensitivity to NUTLC-2, NUTLC-4 and NUTLC-5 tumor cells differed individually according to MTT colorimetric assay, and characteristic drug sensitivity was not noted in histological types of lung cancer.

The authors investigated methods for analysis of oncogenes and tumor suppressor genes in lung cancers and bronchial lesions from high risk patients (retired poison gas factory workers). Amplifications of C-, L-, N-myc, length of terminal repeat array (TRA), mutations of p53 gene, p53 mRNA and K-ras genes were analysed in frozen specimens of surgically resected lung cancers. Various lesions including dysplasia, squamous metaplasia, goblet cell metaplasia, and basal cell hyperplasia were detected in the bronchial epithelium of biopsied specimens from retired poison gas factory workers. Analysis of p53 gene and k-ras gene mutations was performed on these formalin fixed, paraffin embedded samples, but no evidence of mutation has been found to date.

Fluorescent in situ hybridization (FISH) with biotinated chromosome-specific repetitive DNA probes was used for the cytogenetic study of lung tumors and three cell lines of human lung cancer. The authors utilized a set of satellite DNA probes, specific for chromosomes 7, 17, X, Y in order to detect numerical chromosome aberrations in tumor cell nuclei. Normal diploid human lymphocyte nuclei, which served as the control, have have two signal spots in 95% of nuclei in response to 7, 17 chromosome probes. However, lung cancer cells have numerical heterogeneity, and copy numbers as determined by FISH were not definite with each probe. Discrepancies between cytogenetic and flow cytometric studies in the detection of aneuploidy in some tumors were shown. The number of FISH spots showed a correlation only with the Ki-67 labeling index expressed in proliferating cells. Loss of the Y chromosome in a high percentage of cells was seen by FISH in some tumors from male patients. These data indicate that FISH with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of lung tumors with respect to the detection of numerical chromosome abnormalities.

MuLV-integration sites were analyzed on seventeen thymic leukemia cell lines which have been established from spontaneous thymic leukemias in AKR mice and bone marrow chimeras. Three proviral integration sites were identified; near c-myc, N-myc and pim-1. Among them the integrations near the N-myc were analyzed. Two cell lines from AKR and a cell line from [(BALB/c x B6) F1-->AKR] bone marrow chimera contained the proviral integration near N-myc. In all three cell lines the integration of the provirus was found 18 to 20 bp downstream of the translational termination codon. The partial sequence analysis of the integrated LTR cell line established from AKR thymic lymphomas was the same as AKV. In contrast, the LTR integrated in a cell line from a bone marrow chimera was different from that of MuLV so far reported.

We established a new cell line (TC-1) from primary site of a renal cell carcinoma (RCC) patient. Its doubling time in tissue culture was 20 hours at 45th passage and mycoplasma contamination test was negative. The karyotypic analysis demonstrated a human karyotype with a modal number of 70. A consistent chromosomal abnormality was noted such as No. 4 monosomy, No. 7 trisomy and a loss of Y chromosome. Electron microscopic examination showed a brush border, vacuoles and abundant glycogen granules in the cytoplasm, which was compatible with RCC cells. This cell line was transplantable to nude mice and the grown tumor closely resembled the original tumor, i.e. clear cell type and hypervascularity. High titer of interleukin-6 (IL-6) was detected in the supernatant of TC-1 cell culture (approximately 5 ng/ml) as well as in sera of nude mice bearing this tumor (260 pg/ml). Exogenous IL-6 did not enhance the TC-1 cell proliferation as determined by cell count. Flow cytometric analysis could not demonstrate the existence of IL-6 receptor on the cell surface. These results suggested the produced IL-6 did not act as an autocrine growth factor in the cell line. Additional IL-1 alpha to the culture medium induced 3-4 times higher concentration of IL-6 in the culture supernatant compared with that of non-stimulating cells, while exogenous TNF alpha did not stimulate IL-6 production.

Advances in molecular genetics in the past decade enabled us to analyze the cause of mendelian disorders at molecular level and a variety of mutations, not only in point mutations and deletion in exons but also in those occurred in regulatory elements or in RNA processing have been precisely identified. Such a variety of mutations may constitute variable clinical manifestations even in the simple mendelian disorders. On the other hand, pathogenesis of common diseases is much complicated and remains greatly to be elucidated. However, if we could use the strategies applied in the past few years for mendelian disorders, it seems to be not difficult to approach them. It is recommended to categorize a certain disease into subgroups for distinguishing their heterogenous phenotypes by clinical, biochemical and other properties. Owing to the success in making a subgroup (FAB classification), many subtype-specific translocations were found in leukemia, and then, rearrangement of relevant genes is also being shown. The best example is seen in chronic myelocytic leukemia. Since rearrangement of ABL and BCR was shown and both genes were cloned, detection of minimal residual diseases after intensive treatment became possible at 10(-6) level using RT-PCR technique. Recently developed interphase cytogenetics using FISH has visualized Ph1 translocation in metaphase cells and also in round nuclei, suggesting a potential use in monitoring the effect of certain drugs during treatment. Furthermore, very selective targeting therapy is being devised using antisense DNA.