p21WAF1/CIP1 is a potent, tight-binding inhibitor of Cdks which is induced by the wild-type p53 gene. We examined p21WAF1/CIP1 mRNA expression in 16 surgically excised human colorectal tumor and nontumor tissues using Northern blot analysis with reference to the identification of p53 gene mutation. p53 gene mutation was detected in six tumor tissues but not in the other 10 using the PCR-SSCP method. The p21WAF1/CIP1 mRNA level was lower in tumor tissues than in the corresponding nontumor tissues. The mean expression level of p21WAF1/CIP1 mRNA was lower in tissues in which the p53 gene mutation was detected than in those in which it was not, although the difference was not significant. The relative p21WAF1/CIP1 mRNA expression level was significantly higher in the tumors with liver metastases than in those without. These results suggest that the p21WAF1/CIP1 mRNA expression level, which might be a indicator of colorectal cancer malignancy, is suppressed in human colorectal tumor tissues compared with the corresponding nontumor tissues and that the wild-type p53 gene is one of the factors inducing p21WAF1/CIP1 mRNA expression.

p53 status was examined in a total of 52 primary breast cancers, using a combination of yeast-based assay for transcriptional activity of p53 (yeast functional assay, YA) and immunohistochemical staining (IHC) for p53 protein accumulation. Results by the two methods were compared, and their correlations to the clinicopathological characteristics were analyzed. Nineteen cases (37%) were shown to have p53 mutations by YA, while 11 (21%) were shown to be positive of p53 accumulation by IHC. The tumors were classified into 4 groups according to the results by the two methods: Group I (8/52), YA (+) & IHC (+): Group II (11/52), YA (+) & IHC (-): Group III (3/52), YA (-) & IHC (+) and Group IV (30/52), YA (-) & IHC (-). DNA sequence analysis of all the YA (+) cases showed missense mutations in 7 out of 8 cases of Group I and nonsense or frameshift mutations in 8 out of 11 cases of Group II. The presence of p53 mutations significantly correlated with the absence of estrogen receptor in the tumors (Group I + II versus Group III + IV), whereas the number of stained cells positive of p53 did not. Microvascular invasion was significantly more frequent in Group I (75%) than in Group II (27%) (P < 0.05). Survival of the patients assessed by Kaplan-Meier method was the best in Group III, followed by Group IV, II and I (P < 0.05) in that order. These results indicate that tumor p53 status determined by the yeast functional assay and immunohistochemistry can serve as an important prognosticator for patients with breast cancer.

The nm23, encoding a protein identical to nucleoside diphosphate (NDP) kinase, is thought to be a candidate metastasis suppressor gene. Low expression of nm23 at the protein or/and mRNA levels was associated with metastatic potential of several types of rodent and human tumors. Expression and anti-metastatic action of nm23/NDP kinase gene products of human head and neck cancer (squamous cell carcinoma) cell lines were investigated. Among six cell lines examined, only those which showed metastatic potential exhibited reduced levels of nm23-H2 protein with no apparent change in nm23-H1 protein levels as compared with non-metastatic cells. When either nm23-H1 or nm23-H2 cDNA was transfected into a high metastatic cell line, only cell clones expressing higher levels of nm23-H2 protein above control levels displayed a reduced number of lung foci in an experimental metastasis assay. The data suggested that nm23-H2 gene product possesses the metastasis suppressor activity in human head and neck cancer cells.

We evaluated the clinical significance of p53, p27 and PCNA expressions in esophageal squamous cell carcinomas. Operative specimens obtained from 70 patients with esophageal squamous cell carcinomas were investigated by staining with antibodies against p53, p27 and PCNA. The correlations among p53, p27 and PCNA expression, clinicopathologic factors and prognosis were studied. The expression of p53 was observed in 55.7%, and it did not correlate to histologic classification, growth pattern, and lymphatic invasion. The expression of p27 was observed in 48.6%, and it correlated to lymph node metastasis. The PCNA positive index (PCNA-PI) was high in the p27 negative cases. The p53 positive and p27 negative group revealed the worst survival rate among groups. Therefore, the p27 positive case, often associated with well differentiated carcinoma, tends to have a slower tumor-growth rate and better prognosis. It is suggested that immunohistochemical assessment on the p53 and p27 expressions would be useful to the recognize the malignant potential and differentiation of squamous cell carcinoma of the esophagus.

A 53-year-old female case of cytogenetically relapsed chronic myeloid leukemia after allogeneic bone marrow transplantation (BMT) who achieved remission by withdrawal of immunosuppressant is reported. On day 690 of this presentation she is well and alive with performance status of 100%. She had episodes of cyclic oscillation of her neutrophil count during hydroxyurea therapy lasting 1 year before transplantation. Increase of the neutrophils at the time of BMT might have contributed to her early relapse on day 207. Withdrawal of immunosuppressant was successful at least in this case.

Aberrant crypt foci (ACF) are small lesions identifiable in whole-mount preparations of normal-appearing human colonic mucosa with the aid of methyleneblue staining under stereoscopic microscope. Highly frequent mutations of K-ras genes were found, and increased proliferative activities were also observed. Therefore, K-ras gene mutation may have some role in formation of ACF. Using rat experimental model, formation of ACF was shown to be enhanced by cancer promoters (such as secondary bile acids), and to be suppressed by chemopreventive agents (deoxycholic acid and aspirin). Based on these findings, ACF are considered to be precursors of colon cancer. We demonstrated that ACF are the precursors of adenoma-carcinoma sequence. Moreover, we showed that ACF may be suitable biomarkers of chemopreventive agents for colon cancers.

We examined the effect of butyrate on the expression of WAF1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G1-phase arrest. WAF1 mRNA was rapidly induced by treatment with 5.0 mM butyrate, and significant WAF1 protein induction was detected. Using several mutant WAF1 promoter fragments, we found that the butyrate responsive elements are specific Sp1 sites. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1 promoter through specific Sp1 sites in a p53-independent fashion. Based on our results, we propose a novel approach for cancer chemoprevention, which we term "gene-regulating chemoprevention". Our strategy is to activate the potent function of growth-inhibitory genes, which are preserved in cancer cells. WAF1 is a good candidate, because it rarely appears to be mutated in common human tumors, while the p53 gene is frequently mutated. Therefore the p53-independent activation of WAF1 by butyrate could be applied to the prevention of cancer when p53 is mutated.

Among 2087 patients who underwent primary resection of colorectal cancer in our department within the past 20 years, 68 (3.2%) were found to have a secondary or additional cancer in their large bowel after the first resection. Statistically significant risk factors in the secondary cancer group were a high incidence of coexisting adenoma and high frequency of colorectal cancer in second-degree relatives. Those in whom secondary cancer occurred were divided into two groups: the adenoma component (AC) group; and the non-adenoma component (DN) group. Secondary cancer in the DN group revealed lower grade of cell differentiation and more frequent depressed macroscopic findings than in the AC group. As a result, the prognosis of the DN group was worse than that of the AC group (p < 0.05). Based on these results, the recommended follow-up should include colonoscopy in the first years after primary cancer resection and according to the findings of the first colonoscopy the frequency of colonofiberscopic follow-up must be decided.

There are two entities in the predisposition for colorectal cancer. One is the hereditary colorectal cancers which include familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), and others. In the case of these hereditary diseases, genetic testing to detect germline mutation in the causative gene in family members is useful for identifying possible gene carriers. For the diagnosis of HNPCC, the present clinical criteria are not sufficient and analysis of DNA replication error (RER) could be useful. Another entity is genetic sensitivity to environmental carcinogens. In particular, the polymorphism of the genes encoding drug-metabolizing enzymes has been shown to be important in recent years. The fast acetylator genotype of N-acetyltransferase (NAT) genes and the null genotype of the glutathione S-transferase M1 gene have been reported to be associated with increased risk of colon cancer. In the strategy against colon cancer, it is extremely important to identify high-risk individuals so that we can prevent cancers or detect them in earlier stages.

Recent advancement of molecular biology disclose responsible genes of FAP(familial adenomatous polyposis) and HNPCC(hereditary non polyposis colorectal cancer). Gardner Syndrome is now categorized as subtype of FAP. Turcot Syndrome is now known as a heterogeneous disease. Turcot Syndrome caused by APC gene develops medulloblastoma and Turcot Syndrome caused by mismatch repair gene develops glioblastoma. Because of the discovery of APC gene, the presymptomatic diagnosis of asymptomatic gene carriers are now available and preventive surgery can be planned. FAP patients with mutated APC gene between codon 1250 and 1464 shows severe phenotype. It is known that FAP patient whose APC gene mutation locates at codon 1309 develops cancer 10 years earlier in comparison to the rest of the cases. Consequently risky rectal mucosa should be removed in this group of patients. As for HNPCC, presymptomatic diagnosis is still not possible because the penetrance rate has not been estimated yet and some additional responsible genes are expected to be discovered. Replication error, mutator phenotype of mismatch repair gene is useful indicator to predict second primary cancers. When the patient in a HNPCC family develops adenoma with microsatellite mistability, preventive colectomy might be one of the surgical option with the informed consent of the patient.

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. To further understand resistance to topoisomerase (topo) II inhibitors, 50 sublines were isolated as single clones from parental cells by exposure to ETP or m-AMSA. Subsequently, a population of cells from each subline was exposed to three-fold higher drug concentrations allowing 16 stable sublines to be established at higher extracellular drug concentration. The frequency and nature of mutations in topo II in the drug selected cell lines have been evaluated. In order to screen a large number of cell lines, an RNase protection assay was developed. Fragments covering the entire coding sequence of topo II was isolated after PCR amplification and subcloned in pGEM3Z vector. Using this approach, mismatches was observed in 13.6% of resistant cell lines (12% of resistant cell lines exposed to lower drug concentrations and 18.8% of resistant cell lines exposed to higher drug concentrations). Our findings suggest that mutations of topo II gene seem to be an important and frequent mechanism of resistance to topo II inhibitors.

Telomerase, a ribonucleoprotein enzyme which can compensate for the erosion of telomeric repeats each cell division, is considered to be associated with cellular immortality. The genes encoding the major components of human telomerase, TERC for telomerase RNA component, TEP1 for a human homolog of p80, and TERT for catalytic subunit, have been cloned. In addition, TERF1 and TERF2, encoding telomeric repeat binding proteins, have been cloned and shown to be involved in telomere length regulation and the protection of chromosome end fusions, respectively. Although the activity level of telomerase is similar at each stage of the cell cycle in telomerase-positive cells, it is generally upregulated with cellular proliferation and repressed with withdrawal from cell cycle. Although biological mechanisms regulating telomerase activity and telomere length remain elucidated, telomerase activity can be detected in approximately 85% of human malignant neoplasms, and the telomerase is a promising and novel tumor marker for clinical diagnosis of cancer, as well as a novel target of anticancer therapy.

We report six cases of primary pulmonary lymphoma in which a gene-rearranged band was shown by Southern blot analysis of transbronchial or surgical biopsy specimens. The cases were 3 mens and 4 women with an average age of 59.6, who had abnormal pulmonary shadows on chest X-ray. Two cases had Sjögren's syndrome and one case had monoclonal gammopathy (IgM-lambda type). Histopathologic and immunologic examination revealed diffuse infiltration of small lymphocytes (B-cell type) in all cases, and histological features of lymphoepithelial lesion or lympho-cytic interstitial pneumonia (LIP) in three cases. CT revealed an ill-defined margin, air-space consolidation, and air-bronchogram in all cases. A CT-angiogram sign was apparent in 2 cases. In all cases, Southern blot analysis of the tissue obtained from transbronchial or surgical biopsy revealed a gene re-arranged band, supporting the diagnosis of primary pulmonary lymphoma. We infer that an ill-defined margin, air-bronchogram and air space consolidation on CT were associated with the histopathological features of primary pulmonary lymphoma.