Telomerase is a ribonuclear protein that is detected in more than 90% of primary cancer tissues using a telomeric repeat amplification protocol (TRAP) assay, thus, telomerase is considered to be a novel marker for cancer. Telomerase activity is not detectable in somatic cells, except for hematopoietic cells and cryptic cells in the intestine and hair follicles, thus, quantitation of telomerase is important to delineate clinical implication of telomerase activity. We have developed semiquantitative fluorescence-based TRAP assay. Moreover, we also developed an in situ TRAP assay that detects telomerase activity at the cellular level. Thus, using these TRAP assays, we are able to detect telomerase activity in various kinds of extracts or cytological specimens and therefore these applications may have additive information in the early detection of cancer and monitoring disease condition.

Fluorescence in situ hybridization with biotinylated repetitive DNA probe specific for the centromeric region of chromosome 17 (p17H8: Oncor) was applied to suspended nuclei which were isolated by Shutte's method from formalin-fixed paraffin-embedded tissue. The tissues were obtained from surgically resected specimens from nine patients with non-small cell lung carcinoma. The isolated nuclei were prepared with 0.05% pepsin/0.1NHCl for 15 minutes at 37 degrees C. Subsequently, these were immersed in 70% acetic acid for 10 seconds at room temperature. After heat denature with hybridization mixture which contained 3 mu 1 DNA probe for 10 minutesat 70 degrees C, 1 x 10(6) nuclei were incubated overnight at 37 degrees C. After washing with 60% formamide/2 x SSC, the hybridized probes were labeled by FITC conjugated avidin. A number of centromeric signals of chromosome 17 wasevaluated by fluorescence microscopy (BH-2, Olympus). Furthermore, a probe-related FITC intensity was quantified using flow cytometry (FACScan, Becton Dickinson). As the results, there was good correlation between a relative fluorescence intensity determined by flow cytometry and a relative fluorescence signal by fluorescence microscopy (p < 0.05).

Development of novel strategies is required for cancer treatment because most human tumors are refractory to current conventional therapy. During the past decade, a number of oncogenes and tumor suppressor genes have been cloned and the molecular mechanisms as to how mutations in such genes contribute to tumor development are going to be clarified. It is, therefore, a great challenge to develop novel strategies for tumor specific therapy based on molecular biology of cancer. p53 tumor suppressor gene is one of the most frequently mutated genes in a variety of human cancers. This review paper introduces several recent approaches related to the p53 as a molecular target of cancer treatment, including (i) p53 status and chemo-radiosensitivity, (ii) p53 gene therapy, (iii) E1B-deficient adenovirus and (iv) restoration of p53 function by synthetic polypeptides.

Telomerase is a ribonucleoprotein, telomere specific reverse transcriptase, which contains some protein components and telomerase RNA components. Human telomerase RNA and some telomerase components have been identified but not completely. More recently, human telomerase catarytic subunits have been cloned, which are called hTRT or hEST2. The expression of hTRT in human cultured cells is well correlated with telomerase activity and immortality. Moreover, the expression of hTRT in cancer tissues is higher than that of normal tissues. These results suggested that hTRT and telomerase activity may be a powerful additional tool for cancer diagnosis.

This paper deals with the basic features of the xenobiotic efflux pump (P-glycoprotein and MRP) and the clinical significance of the search for specific modulators of these proteins. P-glycoprotein and MRP function as ATP-dependent efflux pumps that extrude cytotoxic drugs from the cells before the drugs reach their intracellular targets, thus conferring resistance to many structurally dissimilar anti-cancer drugs. These proteins are responsible for multidrug resistance of tumor cells, a major obstacle to cancer chemotherapy. To develop well-designed modulators, structural information regarding the specific drug binding sites is important. We recently found that mutations in the putative transmembrane domain (TM) 1 of human P-glycoprotein alter the drug resistance pattern. Some amino acid residues in TM1 together with TM5-6 and TM11-12 may help to govern substrate specificity. The features common to substrates for P-glycoprotein and MRP are also discussed.

Numbers of molecular targets for anticancer agents have been increased during the recent scientific progress in cancer biology. Of these targets, one should understand which target is clinically applicable. The forthcoming approach to evaluate such "molecular targets" is expected to provide novel diagnostic markers for adequate cancer therapy, and also novel targets for development of potent and useful therapeutic agents.

The concept of tumor suppressor gene introduced in the last decade has provided an enormous understanding of the mechanism of human carcinogenesis with the intensive study of p53 during the 1990's. The product of p53 tumor suppressor gene is a transcription factor which recognizes and binds to a specific DNA consensus sequence existing in the promoter region of p53 responsive genes. The main physiological functions of p53 are (1) cell cycle regulation mainly at the G1 check point, (2) induction of apoptosis, and (3) stabilization of genome. As each of them are indispensable gatekeeping devices of the cell for the suppression of malignant transformation, the alteration of p53 gene, which is found more than half of human malignancies, may play a central role in multistep carcinogenesis.

Androgens play a key role in promoting normal sex differentiation and development, pubertal masculinization, initiation of spermatogenesis, and maintenance of male sexual function. In addition to these classical function, androgens are significantly associated with cell differentiation, proliferation as well as carcinogenesis. The function of androgens can be mediated by the androgen receptor (AR), which transduces the steroid signal within cells. AR belongs to the superfamily of nuclear receptors that employ complex genetic mechanisms to control the development and physiological functions of target tissues. AR activates or represses gene transcription through association with specific DNA elements and/or proteins. Moreover, molecular investigations of AR gene structure have provided new insights towards defining a genetic basis for the relationship between the diseased conditions and androgen action. Recent data on androgen biosynthesis, action of androgens and molecular genetic analysis of gene structure have led to a new understanding of the pathology in affected men. This review summarizes briefly our current view of androgen action with a special emphasis on the alterations of AR.

Seventy one patients with renal cell carcinomas were examined for a variety of markers associated with tumor malignancy: nuclear DNA ploidy, AgNORs, PCNA and c-erbB-2. Usefulness of the markers in predicting the prognosis was studied by analyzing the relationship between each of these markers and the prognosis of the patients with renal cell carcinomas. DNA ploidy was analyzed by flow cytometry. AgNORs were stained by the silver colloid method. PCNA and c-erbB-2 were detected by immunohistochemistry. In all the patients examined, DNA ploidy, AgNORs, PCNA and c-erbB-2 were significant predictors of the prognosis. Of the patients with grade 2 carcinomas, the survival rate was significantly higher in the patients with the PCNA-positive cells of lower than 35.0% than in those with the positive cells of more than 35.0%. The patients without the expression of c-erbB-2 exhibited a significantly higher survival rate than those with the expression. In the patients with grade 2 carcinomas, however, neither DNA ploidy nor AgNoRs was a significant predictor of the prognosis. These findings suggest that PCNA and c-erbB-2 provide more accurate information than the others to understand the biological characteristics of the grade 2 carcinomas and are useful in predicting the prognosis of the patients with grade 2 renal cell carcinomas.

Many genetic lesions found in non-small cell lung cancer (NSCLC) have been reported to be associated with a poor prognostic outcome of this disease. Such alterations include mutations of ras genes, overexpression of myc or erbB2 genes and inactivation of RB genes. Among them, the prognostic implications of the p53 gene have been most extensively studied; over 20 reports have been published. However, the significance of the p53 gene abnormality also still remains unclear. Therefore, we re-evaluated our 562 patients with NSCLC retrospectively to see if the p53 gene abnormality really had an effect on patients' survival in this large cohort. There was no effect of p53 gene abnormality on all patients with NSCLC or on those with squamous cell carcinoma, but p53 abnormality was a significant, independent prognostic marker in adenocarcinoma subset. We should plan a clinical trial of postoperative adjuvant therapy for patient with pulmonary adenocarcinoma incorporating information on the p53 gene status to possibly translate these findings into clinical practice.

Whether chemotherapy or surgical resection is primarily selected for lung cancer therapy is determined based on the pathological diagnosis of small cell or non-small cell lung cancer. In future, however, each standard therapy should be established against the respective subgroups of lung cancer, which will be more precisely defined depending on the biological or the molecular basis, like malignant lymphoma. Much evidence is increasing to help understand the mechanisms mediating differences in clinical behavior and neuroendocrine features between small cell and non-small cell lung cancer. Here we showed the experimental models of tissue-specific gene therapy targeting CEA- or Myc-overexpressing lung cancer cell lines.

Recent advances in the molecular biology research have devoted greatly to the clinical applications of genetic informations. Gene diagnosis and therapy is a new medical field to deal with these applications. Nowadays diseases related with gene abnormalities have been shown to reach 8,450, and gene testings to cover all these should be very wide and variable. So that any one of the clinical laboratories in a hospital or even research institutes and laboratory centers should not be able to handle all the testings. A model system to promote gene testings effectively in Japan, which includes a special department in a hospital and limited specifications to the individual laboratories or centers, were presented. In the case of monogenic genetic disorders, gene testing results are useful not only to confirm clinical diagnosis but to predict future development of disorders preclinically and even during the prenatal or pregestational period. Accordingly, these may evoke critical ethical and social problems. Many of common diseases are polygenic in nature and gene testings in these disorders are not directed to the diagnosis but are quite useful to see the individual characteristics of the patients related to the special phenotypes or even the fate of disease process. Acquired malignancies are known to be resulted from a somatic gene mutation and progress by the associations of further abnormalities. Only limited gene testings are already now in use. Wide future development in this field is expected after getting detailed meanings of gene testings in the individual cancers. Gene testings for infectious diseases are known to be quite effective on the early and accurate diagnosis, and many of these are already covered by the health insurance. In conclusion, gene testings are quite important but variable and laboratory personnel should not be able to deal all of these properly. Considering future development of gene therapy, rapid establishment in a hospital of a special department, Department of Gene Diagnosis and Therapy, which is consisted from 3 divisions such as genetic counseling, gene testing and management, and monitoring and adviser of gene therapy, is highly recommended.

Using a reverse transcriptase-polymerase chain reaction (RT-PCR)-based quantitative analysis method, we investigated MDR1 mRNA expression levels in 58 bladder cancer specimens to determine whether MDR1 gene expression was induced or enhanced in bladder cancers during chemotherapy. In bladder cancer specimens which were obtained from patients treated with anticancer drugs, significantly higher expression levels of MDR1 mRNA were observed than in those from patients not treated with any anticancer drugs (p = 0.0134, Mann-Whitney U test). From 14 patients who had bladder cancer, clinical specimens were obtained before and after neoadjuvant intra-arterial chemotherapy. The expression levels of MDR1 mRNA were significantly higher in the post-treatment specimens than in the pre-treatment specimens (p = 0.0298, Wilcoxon signed-rank test). Of these 14 patients, 7 patients exhibited increased levels of MDR1 mRNA expression after chemotherapy. In 6 patients, there were no changes in the MDR1 mRNA expression levels before and after chemotherapy. Only one patient exhibited decreased levels of MDR1 mRNA expression after chemotherapy. No significant correlations were observed, between MDR1 mRNA expression levels and effect of the chemotherapy determined microscopically, dosage of anticancer drugs, or patient outcome. In conclusion, this study indicates that MDR1 gene expression in bladder cancers is induced and enhanced during chemotherapy. This overexpression of the MDR1 gene may contribute to resistance to anticancer drugs after repeated chemotherapy.

A 77-year-old man was admitted because of massive pericardial effusion and cardiac tumor. Cytological examination of the effusion and histological examination of a subcutaneous tumor in the chest wall revealed diffuse large B cell lymphoma. The immunophenotype of tumor cells was CD5+ CD20+ CD22+ CD38+ HLA-DR+ CD19-. Chromosome analysis revealed complex abnormal karyotypes containing t(8;14) (q24;q32). C-myc gene rearrangement was shown by Southern blotting. Chemotherapy with pirarubicin, cyclophosphamide, vincristin, and prednisolone (THP-COP) was not effective for his lymphoma. He suffered from cardiac tamponade and died at 5 months after diagnosis. Autopsy revealed a large cardiac tumor, extensive epicardial infiltration, tiny tumors in the lung and pancreas, but no lymphadenopathy, the combination of which suggested a primary cardiac lymphoma. Immunohistochemistry for p53 protein showed nuclear staining of more than 50% of the lymphoma cells. In situ hybridization for EBER-1 was negative. Rearrangement of c-myc gene and overexpression of p53 protein are usually observed in Burkitt's lymphoma and some cases of high grade lymphomas including AIDS-associated non-Hodgkin lymphomas. In this case the association of these molecular findings and resistance to chemotherapy is suggested.

Correlations between alterations of the p53 gene and clinical features were examined in childhood acute lymphoblastic leukemia (ALL). We analyzed 147 patients and 38 cell lines for p53 mutations within exons 5 to 9 (2 to 11 in some of them) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. p53 gene mutations were found in 3 of 62 (5%) patients at diagnosis, 1 of 14 (7%) patients at relapse, and 13 of 20 (65%) cell lines in T-ALL, 2 of 20 (10%) patients at diagnosis, 4 of 4 (100%) patients at relapse, and 4 of 5 (80%) cell lines in t(1;19)-ALL, 1 of 23 (4%) patients at diagnosis, 2 of 22 (9%) patients at relapse, and 5 of 12 (42%) cell lines in common ALL other than t(1;19) or t(9;22)-ALL and 3 of 3 (100%) patients at diagnosis in B-ALL. In t(1;19)-ALL, p53 gene alterations were associated with a poor prognosis. The patients with p53 mutations had a trend towards poor prognosis in childhood ALL without B-ALL. p53 gene mutation is not always associated with the current prognostic factors. This alteration may become one of the important prognostic factors, if the detection of a small number of the leukemic cells with the p53 gene mutation would be possible.

The p40tax protein of Human T lymphocyte virus type I (HTLV-I) is known to be a transactivator not only for the own viral gene but also for the various cellular genes (host genes), including a number of cytokine genes and genes for cell proliferation. Its tumorigenicity was also reported in vivo, such as developments of mesenchymal tumor, neurofibroma, mammary carcinoma and large granular lymphocytic leukemia, using the transgenic technique. To investigate the tumorigenicity in lymphoid tissues, a series of HTLV-I pX transgenic rats (lck-pX rats) which expressed the pX gene under control of lymphocyte specific protein tyrosine kinase (p56 lck) gene type 1 promoter was produced. In some lines of lck-pX rats, pX mRNA was expressed selectively in the thymus, lymph nodes and spleen as expected. However, pX mRNA expression was also detected in other tissues from other lines of lck-pX rats. Thymic tumor with epithelial markers developed in lck-pX rats as early as 8 weeks of age and high expression of pX mRNA was detected in the tumor tissue. This is the demonstration that HTLV-I pX gene causes thymic tumor in the transgenic rat and an lck-pX rat may be a suitable animal model for elucidating pathogenetic role of HTLV-I pX gene in lymphoid tissue neoplasia.