We investigated the roles of the extracellular matrix, especially of hyaluronan, in cancer metastasis. A positive relationship was shown between hyaluronan synthetic activity and the metastatic ability of cancer cells by manipulating regulation of gene expression of hyaluronan synthases. These techniques may provide future therapeutic methods for cancer metastasis.

To study the relation between the integration of HBV X, S, Pre-S and C genes and the expression of oncogenes/tumor suppressor genes in hepatocellular carcinoma(HCC).

Most testicular germ cell tumors have serological tumor markers such as alpha-fetoprotein (AFP) and human chorionic gonadotropin beta-subunit (beta-HCG). On the other hand, molecular staging of these tumors has not been well established compared to other urogenital malignancies like prostate cancer. Recently, melanoma antigen (MAGE) which is one of the tumor-associated antigens recognized by cytotoxic T lymphocytes (CTL) has been found to be present in a variety of malignant tumor types and normal testis. In addition, Wilms' tumor-associated gene WT1 has been found to be expressed in some testicular cancers. Thus, we examined the expression of these genes in testicular cancer tissues and peripheral blood of cancer-bearing patients using reverse transcription-polymerase chain reaction (RT-PCR). The expression of the MAGE1-3 genes was examined in 34 testicular germ cell tumors (24 seminomas and 10 nonseminomas). Of the seminomas and nonseminomas, 87.5% and 40% were positive for MAGE1, 91.7% and 60% for MAGE2, and 83.3% and 30% for MAGE3, respectively. The expression of the MAGE genes was not correlated with the tumor stage. The expression of the WT1 gene was quantified in 26 testicular germ cell tumors. WT1 was highly expressed in 5 of the 7 high stage cases, but in only 4 of the 19 low stage cases (p < 0.01). The mRNA of these genes could not be detected from the peripheral blood of patients with high stage tumors. These results suggest that MAGE genes may be useful tumor markers for molecular staging of testicular cancer, especially seminoma, and that the WT1 gene may be a tumor marker for high stage testicular germ cell tumors. However, these genes cannot yet be used for molecular staging of testicular germ cell tumors.

The two major methods of treatment of chronic myelogenous leukemia (CML) are bone marrow transplantation (BMT) and interferon (IFN) administration. BMT is recommended when patients are young (usually younger than 45 or 50 years old) and have an HLA identical sibling. Otherwise, IFN administration is the treatment of choice. IFN administration results in the eradication or reduction of the Philadelphia chromosome. Such cytogenetic effects have brought about an improvement in the survival of CML patients.

It is impossible to predict malignant potential of thymomas by conventional histopathological examination. In order to find a malignant marker of thymoma, we immunohistochemically examined the expression of the products of p53 and p27kip1, potential tumor suppressor genes in thymic epithelial tumors. The thymic epithelial tumors examined in the present study included 13 non-invasive thymomas, 7 invasive thymomas, and 4 thymic carcinomas. The thymic epithelial cells showed abnormal accumulation of p53 protein in 2 of 13 non-invasive thymomas (15.4%), 4 of 7 invasive thymomas (50%), and 3 of 4 thymic carcinomas (75%). The frequency of p53-expression paralleled with clinical aggressiveness. On the other hand, p27 showed no correlation with clinical aggressiveness. In conclusion, the present results suggest that the presence of p53-positive epithelial cells might be a useful indicator to predict malignant potential of thymoma.

Neurofibromatosis type 2 (NF2) is an autosomally inherited disorder, caused by a mutation in NF2 tumor suppressor gene on chromosome 22q12, being characterized by multiple intracranial tumors including schwannomas, meningiomas and ependymomas. The protein encoded by the NF2 gene has a similarity to ezrin, radixin and moesin (ERM) proteins that link membrane proteins to the cytoskeleton. It has been reported that the majority of NF2 gene mutations are nonsense mutations that result in a premature termination of translation. We have developed and established a yeast-based stop codon assay for detection of NF2 gene premature terminating mutations. This assay utilizes an autonomously replicating yeast vector that expresses an NF2::ADE2 chimera protein, which gives a normal white colony when a sample NF2 cDNA is homologously recombinated, while it gives a red colony when the sample cDNA contains a nonsense mutation. The assay gave 8.0 +/- 3.5 (mean +/- SD) background red colonies when tested on clinical samples which did not contain an NF2 gene mutation. A total of 16 schwannomas (including three NF2 cases) were tested by the assay. NF2 gene mutations were detected as red colonies more than 10% in 13 of the 16 cases. Sequence analyses of plasmids recovered from the red colonies showed single base substitutions giving stop codons in five cases, and base deletions leading to frameshift and premature termination in four. Additionally, in-frame exon skippings were found in three cases. One case that gave 14% red colonies did not show a clonal mutation. This study demonstrates that the newly established assay is capable of an efficient detection of nonsense mutations of NF2 gene in clinical samples.

Since homeobox-containing genes (HOX genes) are a family of transcriptional regulators, which give cells positional information in morphogenesis, cancer metastasis can be explained as a heterotopic expression of HOX genes. In the present study, I transfected HOXD3 gene into human lung cancer A549 cells and investigated alterations of adhesiveness, migration and invasiveness of the tumor cells. Overexpression of the HOXD3 gene enhanced expressions of integrin alpha 3, alpha 4 and beta 3 subunits, and increased adhesive and migratory activities toward fibronectin and vitronectin. It was suggested that the increased migration of the tumor cells resulted from enhanced expression and activation of integrin alpha v beta 3. Furthermore, the overexpression of HOXD3 increased mRNA expressions of urokinase-type plasminogen activator and transcription factors ets-1 and -2. Most of these molecules, which increased with overexpression of HOXD3, are well-known factors associated with tumor invasion and metastasis. Indeed, HOXD3 transfectants revealed high invasive activity to matrigel, a basement membrane model, compared to their parent cells and control neo-transfectants. These findings suggest that abnormal expression of HOXD3 may enhance tumor invasion and metastasis through increased expressions of metastasis-related genes.

Metabolism of most chemical carcinogens in humans is thought to occur in two distinct phases. The carcinogens exert their effect only after being metabolically activated to intermediates (phase I), which are capable of binding to DNA and causing mutations. The most ubiquitous phase I catalysts are the cytochrome P450s (CYPs). There is convincing epidemiological evidence that lung cancer risk associated with polycyclic aromatic hydrocarbons (PAHs) is mediated in part by aryl hydrocarbon hydroxylase (AHH), which is used as an indicator of the phenotype of CYP1A1. Since AHH is responsible for the activation PAHs in cigarette smoke, it may be important in the causation of lung cancer. Kellermann et al. reported a significant positive correlation between AHH inducibility and susceptibility to lung cancer. The finding, however, has been both supported and refuted by subsequent investigators. Advances in molecular biology have allowed many allelic variants of several drug metabolizing enzymes so that individuals with the susceptible genotypes can be determined easily. A close association between development of lung cancer and homozygous rare genotypes in MspI and Ile-Val polymorphisms of CYP1A1 gene has been recently reported in some Japanese populations. The association between GSTM1 polymorphism and lung cancer risk is not so strong, however. Following the phase I reaction, phase II enzymes such as glutathione S-transferases (GSTs) are responsible for detoxification of activated forms PAH-epoxides. GSTs form a superfamily of genes consisting of four distinct families, named Alpha, Mu, Pi and Theta. The GSTM1, GSTT1 and GSTP1 genes are polymorphic in humans. The phenotypic absence of GSTM1 activity is due to a homozygous inherited deletion of the gene, the null genotype. The homozygous deletion of GSTM1 genes has been shown to occur in approximately 50% of the populations of various ethnic origins. The GSTM1 null genotype confrs a moderately increased risk of smoking-related lung cancer, however. Genetically determined susceptibility to lung cancer may depend on the metabolic balance between phase I and phase II enzymes. Risk of lung cancer, especially squamous cell carcinoma is shown to be remarkably increased in individuals with a combination of a homozygous rare allele of the CYP1A1 gene and the nulled GSTM1 gene, compared with those having other combinations of genotypes. Individuals with susceptible genotypes may become important for the prevention of lung cancer.

We report on a 16-year-old boy with B cell acute lymphocytic leukemia presenting marked leukocytosis (388,000/microliter) and resistance to multidrug chemotherapy. Karyotypical analysis revealed a novel t(3;15)(q27;q2?2) chromosomal abnormality. Because 3q27 is known to be a locus of the bcl-6 gene, which is frequently involved in B cell malignancies, molecular biological analyses were performed. Although no rearrangement was detected in 5 genes including bcl-6 on 3q27 and 2 genes on 15q2, reverse transcriptase-polymerase chain reaction procedures detected relatively strong mRNA expression of the bcl-6, smrp, dvl3, and tpml genes. These results indicate that immature leukemic cells with CD10 and CD34 positivity and rearrangement of the T cell receptor beta gene may coexist with relatively mature subpopulations that are positive for CD19 and CD20 surface markers, bcl-6 expression, and rearrangement of the gene for immunoglobulin kappa.

Primary splenic lymphoma (PSL) is extremely rare, accounting for less than 1% of all reported cases of extranodal lymphoma. A 62-year-old woman was referred to our hospital because of general fatigue. A heterogenous mass with irregular margins was detected in the spleen by abdominal computed tomographic scan, and Gallium scintigraphy demonstrated abnormal accumulation only in the spleen. Malignant lymphoma was strongly suspected on the basis of histologic findings from an ultrasonically guided needle biopsy. The final diagnosis was established by splenectomy as PSL of diffuse large B-cell type. After 6 courses of CHOP chemotherapy, the patient recovered and has been disease-free more than a year. Chromosomal analysis of her tumor cells detected t(3;14)(q27;q32), an abnormality not reported in cases of PSL to date. The rearrangement of BCL-6 was also observed. We discuss the possibility of BCL-6 involvement in Japanese cases of PSL, with reference to case reports dating back over the past decade.

Mice have widely been used as an experimental model for carcinogenesis induced by chemicals or irradiation. Recently, transgenic mice expressing oncogene proteins and knockout mice lacking for tumor suppressor genes are available, and used for the analysis of mechanisms underlying carcinogenesis. In such experimental carcinogenesis, a rapid and sensitive method for screening p53 mutations is desired. In human carcinogenesis, p53 yeast functional assay has been proved to be a very useful method for screening p53 mutations. However, the p53 yeast functional assay has been unsuccessful in mice because of the high background mutations in them. In the present study, the author developed a mouse p53 yeast functional assay by reducing the background mutations. Initially, 25.8 +/- 2.8% of background mutant red colonies were given by total RNA from normal mouse liver. The background level was lowered to 13.6 +/- 3.3% by improvement of RT-PCR conditions. Then the p53 cDNA-containing plasmids were rescued from the red colonies and the cDNA sequences were determined. The analysis revealed that many background mutations were caused by insertions of extra adenine (A) and thymine (T) at A and T homopolymeric runs, respectively. Majority of the insertion mutations occurred at 5' terminus of murine p53 cDNA. Based on these findings, we constructed a new vector and designed an optimal PCR primer set to exclude 5' terminal sequence in the yeast assay. Finally, the background mutation rate was reduced to 8.0 +/- 1.4%, which was comparable with the rate of 5.2 +/- 2.7% in human p53 yeast functional assay. Using the murine p53 yeast functional assay, we screened several cell lines for p53 mutations and determined those mutations by DNA sequencing. Furthermore, we investigated p53 mutations in UV-irradiated skins of XPC-gene-knockout mice. The yeast functional assay followed by DNA sequencing analysis revealed predominant mutations in dipyrimidines in the p53 coding sequence. These results indicate that mouse p53 yeast functional assay will be very useful for the analysis of p53 mutations in experimental carcinogenesis.