Retinoids, all-trans-retinoic acid (AT-RA) and its bioisters, exert diverse and profound effects on the growth and differentiation of normal and malignant cells, vertebrate development, and homeostasis. Since their nuclear receptors (RARs: alpha, beta and gamma) were cloned, extensive studies on retinoid signalling at molecular level have elucidated its elaborate mechanisms, by which the expression of a variety of retinoid responsive genes are regulated. Furthermore, discoveries of the second receptor system of retinoid (RXRs: alpha, beta and gamma) and their substantial ligand, 9-cis-retinoic acid (9C-RA), were recently reported. The heterodimer formation between RXR and RAR or other members of the steroid/thyroid hormone receptor superfamily gave us a new aspect of hormonal gene control including retinoids, thyroid hormone, vitamin D3 and others. These heterodimers were showed to bind specific response elements consisted of the direct repeat of consensus sequence (AGATTC) or its related sequences. Differential regulation of retinoid responsive gene by RARs or/and RXRs in vivo is an ongoing subject and the combinatory outcome of subtypes or isotypes of receptors, natural response elements, natural ligands (AT-RA, 9C-RA, or their metabolites) owing to pleiotropic effects of retinoids is being revealed. The anti-malignant effect of retinoids is another important theme. The report of prominent therapeutic effect of AT-RA against APL patients was sensational. In many APL patients, translocation between RAR alpha gene and an oncogenic gene, PML, can be detected, although the roles of their fusion products (RAR alpha-PML and PML-RAR alpha) in APL induction have not been understood. Furthermore, the cross-talk between RAR and AP-1 is thought to give an explanation for the suppressive effects of retinoids against tumor promoters (TPA and others). In those contexts, synthetic retinoids specific for each subtype of RAR or RXR, which will be useful reagents for biological studies and/or excellent therapeutic agents, are being developed. Some RAR subtype-selective compounds including antagonists have been already reported.

Multidrug resistance to anticancer drugs proved to be related to the MDR1 gene which encodes the P-glycoprotein, an energy-dependent drug-efflux pump for lipophilic drugs. We investigated the expression of the MDR1 gene in clinical samples by RT-PCR. The subjects were all resected cases of 14 colorectal cancers, five gastric cancers, two esophageal cancers, two gallbladder cancers and 20 lung cancers. Adrenal gland was used as a positive control. Total RNA was extracted from a fresh tissue sample. The cDNA was synthesized from 1 microgram of total RNA using reverse transcriptase. With the above cDNA as the template, amplification of the 157-bp fragment of the MDR1 gene was performed using PCR. The PCR product was polyacrylamide gel-electrophoresed and ethidium bromide-stained. In addition, a dot blot analysis was performed to quantify the amount of PCR product. Since PCR was performed simultaneously under the same conditions, the PCR product was quantified at four stages, from (3+) to (-), to indicate the degree of expression of MDR1 mRNA. Adrenal gland showed (3+)-(2+) and colorectal cancer exhibited mostly (2+)-(1+). Both cancerous and non-cancerous areas evidenced a similar degree of expression in the cases of colorectal cancer. The MDR1 gene was expressed at low levels in other digestive tract cancers and in lung cancer. The levels of MDR1 expression revealed no correlation to either histological type or clinical stage. The present method may contribute to designing anti-cancer protocols.

DNA content analysis using flow cytometry and amplification of c-myc, L-myc, and c-erbB-2 oncogenes in 143 cases of resected lung cancer were analyzed using the same specimen, and we examined the correlation with prognosis of DNA content and amplification of oncogenes. There were 54 DNA diploid cases (38%), 81 DNA aneuploid cases (57%) and 8 DNA multiploid cases. Analysis of oncogene amplification revealed 22 cases of c-myc, 4 cases of L-myc, and 22 cases of c-erbB-2. In curatively resected cases, the 5-year survival rate was 65% in 31 DNA diploid cases, and 36% in 40 DNA aneuploid cases. There was a statistically significant difference between the two groups (p < 0.02). However, in non-curatively resected cases, the 5-year survival rate was 11% in 23 DNA diploid cases, and 33% in 49 DNA aneuploid cases. There were no statistically significant differences among these groups. The correlation between DNA content and amplification of oncogenes was as follows. In DNA diploid cases, there were 4 cases of c-myc, and 6 cases of c-erbB-2. In DNA aneuploid cases, there were 15 cases of c-myc, 4 cases of L-myc, and 15 cases of c-erbB-2. In DNA multiploid cases, there were 3 cases of c-myc, and 1 cases of c-erbB-2. Amplification of oncogenes was seen more frequently in DNA aneuploid and multiploid cases than in DNA diploid cases. In 71 curative resected cases, the 5-year survival rate for amplified cases of c-myc (10 cases) was 0%, and that of cases with no amplification was 61% (no statistically significant difference). The 5-year survival rate for amplified cases of c-erbB-2 (10 cases) was 40%, against 52% for cases with no amplification. DNA content analysis using flow cytometry was more convenient than analysis of amplification of oncogenes, and reflects the prognosis of resected lung cancer better than oncogenes. There was no relation between DNA content and gene amplification.

The authors attempted to estimate the relationship between three biological parameters (nuclear DNA content, PCNA (proliferating cell nuclear antigen)/cyclin, HER-2/neu oncoprotein) and lymph node metastasis. We evaluated 37 breast cancers which were less than 2 cm in maximum dimension. Quantitative analysis was performed using a CAS 200 Image Analysis System, after Feulgen staining and immunochemical staining using anti-PCNA/cyclin monoclonal antibody and anti-HER-2/neu oncoprotein polyclonal antibody. In lymph node-negative cases 20.0% were aneuploid, while in lymph node-positive cases 58.8% were aneuploid. A total of 20.0% lymph node-negative cases were in the high proliferation group, as opposed to 52.9% of lymph node-positive cases. This analysis revealed a significant relationship between cell proliferation and lymph node metastasis. Analysis of the expression of HER-2/neu oncoprotein revealed no significant relationship between overexpression of HER-2/neu oncoprotein and lymph node metastasis, but the expression of HER-2/neu oncoprotein was significantly related to a shorter relapse-free survival.

One-hundred and sixty-four patients with gastric carcinomas, who underwent gastrectomy during 1979-1985, were studied. Sixty-five of these cases were early gastric carcinomas, and the others were advanced gastric carcinomas, and the others were advanced gastric carcinomas. The nuclear DNA contents were measured by cytofluorometry, and immunohistochemical study on the expression of c-erbB-2 protein was performed using a monoclonal antibody against the c-erbB-2 oncogene product. Furthermore, immunohistochemical detection of proliferating cell nuclear antigen (PCNA) was performed using a monoclonal antibody against the PCNA. The rates of positive invasion beyond submucosal layer, lymphatic invasion, and vascular invasion in aneuploid cases were significantly higher than those in diploid ones, and the patients with aneuploid tumor had a significantly worse prognosis than those with diploid tumor. The rates of positive lymph node metastases and invasion beyond submucosal layer in the group with positive staining of the c-erbB-2 protein was significantly higher than in the negative group, and the group with positive staining for c-erbB-2 had a significantly worse prognosis than the negative one. PCNA indices showed a significant correlation with lymph node metastasis, and the group with higher PCNA indices had a worse prognosis. The patients with tumor showing both aneuploid and positive staining for c-erbB-2 protein, had the worst prognosis. There is a relationship between c-erbB-2 tissue status and PCNA indices, but no correlations were found among c-erbB-2 tissue status, PCNA indices and DNA contents. From these results, it can be concluded that DNA ploidy, c-erbB-2 protein, and PCNA may reflect the malignant potential of gastric carcinoma.

In order to elucidate the biological characteristics of colorectal cancer invading the proper muscle, we analyzed DNA ploidy and PCNA labeling index. The growth patterns were divided into three types; those accompanied by intramucosal polypoid growth (PG+), those with non-polypoid growth (PG-), and those ranging between PG+ and PG- (PG+/-). The relationship of DNA ploidy to lymphatic vessel permeation and recurrence was also discussed. The results were as follows: 1) Growth types bore no relation to DNA ploidy and distribution of PCNA. 2) In aneuploidy cancer, it showed a high incidence of lymphatic vessel permeation. A trend of lymphatic metastasis was also observed. Aneuploidy cancer showed a poor prognosis compared with diploidy cancer.

One of 8 to 12 pre-B ALL cells co-express CD13 and CD33 antigens, but such blasts do not express myeloperoxidase (MPO) even on electronmicroscopy or mRNA. MPO+ pre-B ALL is extremely rare (1/50-1/100), however a cell-line (Tahr87) was established in culture. In contrast, T-lineage blasts express CD13/33 antigens regularly in the pro-thymic stage (CD7+ 5+ 2+ 3- 4- 8- or more immature), and a limited expression of MPO is rather commonly detected particularly in recurrences. The co-expression of CD3 epsilon/MPO or CD3 epsilon/delta/MPO mRNA has been demonstrated. Thus, the regulation of MPO expression is of utmost importance in interpreting the phenotypes of leukemia/lymphoma. While testing the effects of several cytokines on MPO expression, IFN-gamma was found to suppress the gene expression of MPO in HL60 cells. This suppression was not accompanied by differentiation, termination of proliferation or reduction of cytochemical MPO+ cells, and was reversible. Among 22 cases of M1 AML blasts, 8 cases were HLA-DR(-). DR antigen was induced by the presence of a mixture of IFN-gamma, TNF-alpha and TPA in 4 cases, but not in the other 4 cases. The blasts of the latter 4 cases were always CD34(-), CD7(-) and CD45RA-/RO+, and constituted a distinct M1 subset which has not previously been reported.

A simple one-step double staining with DAPI and hematoporphyrin (HP) was used for UV-microspectrophotometric (UV-MSPM) and flow cytometry (FCM) analysis of the same samples. The specimens analyzed included fresh biopsy and/or surgical materials, trimmed 50 microns-thick paraffin sections and smears (with the latter not available for FCM). We provided the special technology for preserving the cytoplasm as far as possible to permit measurement of cell size and N/C ratio. DAPI indicates DNA content under 365 nm and HP of total protein content under 670 nm UV-MSPM. The DAPI can measure the nuclear size and the latter the cell size. DAPI/HP staining yielded much more accurate measurements than staining with FITC and PI. The use of both MSPM and FCM with cell sorting on the same samples is very helpful in cytology and histopathology for evaluating and differentiating borderline lesions and grade of malignancy, as well as oncostatic effectivity. Our goal is to combine this technology with the automated cytologic screening system CYBEST, which was developed by Tanaka et al, based on a morphometric device.

A case of metachronous bilateral testicular seminomas on a 36-year-old male is reported. He had a history of right testicular typical seminoma treated with right high orchiectomy and adjuvant irradiation therapy 6 years previously. He returned to our clinic complaining of painless left testicular enlargement in August 1990, and under a diagnosis of testicular neoplasm left high orchiectomy was performed. Histopathological examination revealed anaplastic seminoma. DNA histograms of both testicles revealed almost the same aneuploid pattern. The patient was diagnosed as stage I seminoma, has been followed up closely. Recently genetic factors as human leukocyte antigens (HLA)-A24, B14, DR5 and DR7, have been shown to be important in the development of metachronous bilateral testicular tumors. HLA antigens were determined in our case, and HLA-A24 shown in our case was compatible with the review.

Molecular events associated with the transformation into blast crisis phase in Ph1-positive CML were analyzed in the present study. The 9;22 chromosomal translocation in CML generates the bcr/abl fused gene coding P210bcr/abl that has enhanced tyrosine kinase activity. In 55 CML cases, Southern and RT-PCR analysis revealed that breakpoints of the bcr gene on chromosome 22q11 were clustered in M-bcr, except for one case and no obvious difference was observed between chronic and crisis phases. However, blast crisis cells displayed enhanced the expression of bcr/abl mRNA, when compared with those in chronic phase cells. By DNA transfection and PCR analysis, the point-mutational activation of N-ras oncogene was rarely identified, and no point-mutational activation of fms gene was found in the crisis phase cases. On the other hand, 2 out of 13 crisis cases contained gross alteration of p53 anti-oncogene. Furthermore, all 4 myeloid crisis cases and K562 cells showed disappearance of the P53 transcript, and MC3 cells derived from a myeloid crisis case showed an aberrant transcript, whereas chronic phase cases, Ph1-positive ALL cell lines and lymphoid crisis cases including NALM-1 cells showed normal expression of the P53 gene. At present, the precise mechanism associated with the blastic trans-formation in CML remain to be determined. The present study suggested one possibility that a selective and progressive process of Ph1 clone with high expression of the bcr/abl gene may be involved with the transformation into non-lymphoid crisis phases from chronic phases. In addition, this progression may be accelerated by the alteration of p53 anti-oncogene, or/and rarely by the point-mutational activation of ras oncogene family.

Expression of p53 tumor suppressor gene was examined in oral squamous cell carcinomas (OSCC), the precancerous lesions (leukoplakia) and cell lines established from OSCC, to investigate (1) whether or not all p53 mutations can be detected immunohistochemically and (2) when the alterations of the p53 gene and/or protein occur during the genesis of OSCC from leukoplakia. Over-expression was detected in 11 out of 16 cell lines with the anti-p53 monoclonal antibody PAb1801, although the remaining 5 cell lines have previously been reported to have mutations, thus suggesting that the immunohistochemical method has a limitation. To answer the second question, the ZA case was chosen as this case of OSCC with a point mutation at codon 279 had a history of leukoplakias which showed a malignant progression. The polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP) method was employed to determine the presence of the mutation. Immunohistochemistry showed over-expression of the p53 protein in all 5 biopsy materials of leukoplakia, while the mutation was detected in 2 materials. In other 15 cases of leukoplakia, over-expression has a relation to epithelial dysplasia. These results indicate alterations of p53 gene and/or protein are observed not only in OSCC but also in leukoplakia and play an important role in the genesis of OSCC.

A 26 year old Japanese male who had a history of leukocytosis in 1985 and received chemotherapy because of myeloblastic crisis of chronic myelogenous leukemia (CML) from May 1986, was admitted in November 1987. He had lymphadenopathy, lymphoid tumor of paranasal sinus and pleural effusion with marked lymphoid cells infiltration. On admission, laboratory data of peripheral blood and bone marrow revealed remission; lymphoid cells of pleural effusion were positive for CD3, CD4 and CD8. Second induction chemotherapy was performed successfully. After a few months, however, myeloblastic crisis recurred. Intensive chemotherapy ended in failure and he died of renal and heart failure. Chromosome analysis showed Ph1 and additional abnormalities at myeloblastic crisis and normal at T lymphoid crisis, but the same rearrangement of breakpoint cluster region existed in both crisis cells. Therefore we supposed that more than two-step pathogenesis is involved in the development of Ph1 positive or Ph1 negative CML clone of this patient.

We attempted to identify the cytogenetic significance in predicting the prognosis of patients with myelodysplastic syndrome (MDS). From the results, we established scoring system (Bournemouth scoring+cytogenetic scoring: 3 for > or = 3 chromosomal abnormalities; 1 for -7/7q-, +8, 2 abnormalities); patients with score of > or = 5 had significant worse prognosis than that with score 0-2. Moreover, RARS with complex abnormalities consists of specific subtype showing a high leukemic rate with a high mortality rate. MDS with hypocellular marrow showed prognosis similar to those with typical MDS, while those with minimal dysplasia had prognosis similar to those with aplastic anemia. Patients with MDS were categorized into 3 subtypes, i.e., early disease-evolution, late disease-evolution, and no disease-evolution group. About 50% of patients with complex abnormalities showed early disease-evolution, while the remaining died before disease-evolution. In patients showing late disease-evolution, no cytogenetic factor are informative to predict the timing of disease-evolution, except percentage of marrow blasts at the diagnosis.

Recent advances in molecular cytogenetics of leukemia is reported with special reference to the pathogenesis, diagnosis, prognosis, and potential gene therapy. Regarding leukemogenesis, we found that neocarzinostatin induced a variety of deletions and reciprocal translocations. Among these random chromosome abnormalities, two reciprocal translocations which were specific for certain leukemias could be observed; t(11;14)(q13;q32) and t(7;11)(p15p13). This fact suggests that a translocation carrying oncogene rearrangement may be of potential relevance to the leukemogenesis. The success in making a subgroup (FAB classification) identified a number of subtype-specific translocations in leukemias. It has been suggested that an initiation or progression-associated event is mediated through a gross chromosomal change. The molecular characterization of chromosomal rearrangement leads to the identification of genes involved in leukemia. Our recent works in molecular cytogenetics of chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), FAB-M3 and -M4 were shown in this article. Since rearrangement of relevant genes were cloned, PCR made it feasible to detect minimal residual disease at 10(-6) level after intensive treatment or bone marrow transplantation for CML, Ph-positive ALL, M3 and approximately half of childhood leukemia. Recently developed fluorescent in situ hybridization (FISH) using specific probes can visualize certain chromosomes or chromosomal segments. Ph translocation, for instance, is now demonstrated as three spot-signals in interphase nuclei using YAC (yeast artificial chromosome)-BCR clone. Lastly, the use of antisense oligonucleotides for the BCR-ABL junctions should result in the inhibition of growth of CML clone. The strategy using antisense molecules may be very powerful tool in the gene-targeting therapy for human neoplasms.

The usefulness of the measurement of nuclear DNA content by fine needle aspiration biopsy was evaluated through a comparative study of fine needle aspirated and tissue samples. Nuclear DNA content was measured in patients with 28 primary and 2 metastatic lesions of breast carcinoma using fluorescent cytophotometry with Feulgen stain. The rate of correspondence on DNA ploidy pattern of aspirates and tissue samples was 86.7%. There was a significant correlation with DNA index (DI) (R = 0.85, p < 0.001) and S-phase fraction (%S) (R = 0.79, p < 0.001). The rate of correspondence of DI subgroups (3 aneuploid subgroups and diploid) was 80.8%. No tumor size affected values of DI and %S of aspirates. However, %S of papillotubular carcinoma showed small difference compared with values of tissue samples. Nuclear DNA content of fine needle aspirated samples had a significant correlation with that of tissue samples, providing the estimation of the original ploidy pattern and DI. Fine needle aspiration biopsy is an easy technique and able to be carried out repeatedly. Thus, the measurement of nuclear DNA content using aspirates is useful for broad practical purposes.

Tumor cells derived from a metastatic mediastinal lymph node of a thirty six year old man with large cell lung cancer were transplanted into the subcapsule of the kidney of a nude mouse. The growing tumor was transplanted into a nude mouse twice and, thereafter, cultured in vitro. After the twelfth passage in vitro, the cell (NUTIY) was transplanted into subcutaneous tissue of a nude mouse and formed a rapidly growing tumor, showing the histological pattern similar to the large cell lung cancer of the patient. However, the chromosomal analysis revealed only murine chromosomes for NUTIY and any transfected human genes were not found in the genes of NUTIY by means of Alu probe. H-ras oncogene of NUTIY was amplified 4 folds, but that of the primary lung cancer and the metastatic lymph node of the patient was on a level with normal cells. NUTIY might be a cancer cell which a murine cell had been transformed into by some carcinogen in vitro.

FISH method with chromosome specific DNA probe to interphase nuclei has been useful for the analysis of chromosomal numerical aberration in human brain tumor. We applied the FISH method to 9 gliomas and one glioma cell line (KMU-100) with 4 kinds of chromosomes of number 7, 9, 10 and 17. The predominant specific aberration in glioma were revealed the increased signal numbers of chromosome 7&17 and the decreased signal numbers of chromosome 9&10. The inactivation of tumor suppressor genes and the activation of oncogenes have been suggested as the principal mechanism of tumorigenesis in human cancer. The definite oncogenes have not still been identified on chromosome 7&17 and tumor suppressor genes on chromosome 9&10. We discussed the mechanism of tumorigenesis with one or more oncogenes on chromosome 7&17 and of one or more tumor suppressor genes on chromosome 9&10 in glioma.

Primary diffuse infiltrative cancer of the large bowel shows poor prognosis. A human rectal cancer cell line, designated as SRM, was established from the metastatic lymph node of a 35-year-old female patient. SRM cells have been cultured with RPMI-1640 medium supplemented with 10% fetal calf serum and grew as monolayers, showing a tendency to pile up. The doubling time of this cell line was 23.0 hours, and the modal number of chromosomes was 64 at passage 14. The cells produced CA19-9 and TPA in the spent medium and formed tumors in nude mice, the histology of which was similar to that of the primary tumor. CA19-9 in the cytoplasma of the transplanted tumor cells was demonstrated by the ABC method and the c-myc oncogene was amplified in the transplanted tumor in nude mice.