Soichiro Torigoe

Retinoblastoma gene mutations in primary human bladder cancer.

Inactivation of the retinoblastoma (RB) gene is known to be implicated in the pathogenesis of several types of human cancers. Since structural alterations of the RB gene have not been well examined in human bladder cancer, we looked for mutations in the entire coding region of this gene using polymerase chain reaction (PCR) and single-strand conformational polymorphism analysis of RNA. We also examined allelic loss of the RB gene using PCR-based restriction fragment length polymorphism analysis. Of 30 samples obtained from patients with bladder cancer, eight (27%) were found to have RB gene mutations. DNA sequencing of the PCR products revealed five cases with single point mutations and three cases with small deletions. These mutations included one (10%) of ten low-grade (grade 1) tumours, four (50%) of eight intermediate-grade (grade 2) tumours and three (25%) of 12 high-grade (grade 3) tumours. Likewise, mutations were found in four (21%) of 19 superficial (pTa and pT1) tumours and four (36%) of 11 invasive (pT2 or greater) tumours. In 15 informative cases, loss of heterozygosity at the RB locus was shown in five cases (33%), three cases with RB mutations and two without them. These results suggest that RB gene mutations are involved in low-grade and superficial bladder cancers as well as in high-grade and invasive cancers.

SN-38, a metabolite of the camptothecin derivative CPT-11, potentiates the cytotoxic effect of radiation in human colon adenocarcinoma cells grown as spheroids

BACKGROUND AND PURPOSE CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is anew semisynthesized derivative of camptothecin. SN-38 (7-ethyl-10-hydroxycamptothecin), a metabolite of CPT-11, plays a key role in the action of CPT-11. MATERIALS AND METHODS To determine whether SN-38 potentiates the cytotoxic effect of radiation, we investigated the interaction of SN-38 and radiation in vitro in monolayer cultures and multicellular spheroids of HT-29 human colon adenocarcinoma cells. RESULTS HT-29 spheroids were more resistant to both SN-38 and irradiation than monolayer cells. SN-38 at a concentration of 2.5 microg/ml, which by itself was not cytotoxic, greatly increased the lethal effects of radiation in spheroids, but not in monolayer cultures. Exposure to SN-38 following irradiation inhibited the potentially lethal damage repair (PLDR) in spheroids. It is suggested that the mechanism of the radiosensitization by SN-38 is due to the PLDR inhibition. CONCLUSIONS These results indicate that CPT-11 may play a role as radiosensitizer and that a combination of CPT-11 and irradiation could prove to be a particularly effective strategy with which to treat human colon adenocarcinoma.

Expression of transforming growth factor-beta 1 in human bladder cancer

Background. Elevated expression of transforming growth factor‐beta 1 (TGF‐01) has been reported in several types of human cancer. However, the significance of TGF‐β1 expression in clinical bladder cancer is not well known.

Polymorphisms in the human DNA polymerase β gene

Recently, evidence has accumulated that mutations in DNA repair genes might be associated with certain steps in carcinogenesis. The DNA polymerase β gene is one of the DNA repair genes, and mutations in it have been detected in 83% of human colorectal cancers. To assess the involvement of polymerase β gene mutations in the development of human prostate cancers, we performed sequence analyses of human DNA samples. Unexpectedly, we found six regions that were polymorphic. This information should be taken into consideration at the time of sequence analysis of the DNA polymerase β gene.s

Molecular genetic diagnosis of von Hippel-Lindau disease : analysis of five Japanese families

We analyzed deoxyribonucleic acids from blood samples of five Japanese von Hippel‐Lindau (VHL) disease families (three familial cases, two new mutations) for the presence of VHL gene mutations by single‐strand conformational polymorphism analysis and direct sequencing. Four of the five families showed germ line mutations in VHL gene, comprising 2 missense mutations, 1 deletion, and 1 splice‐site mutation. Two families had VHL gene mutations at exon 1; 1 family at exon 3; and 1 family at the splice‐site adjacent to exon 3. Presymptomatic patients were accurately diagnosed by these methods. However, one family did not show a VHL gene mutation in the germ line but showed a somatic mutation at exon 2 in the hemangioblastoma tissue. The consequence of the somatic mutation was a microdeletion leading to a frameshift mutation. Our study is the first report of VHL gene analyses of Japanese VHL disease families, and suggests that not only germ line mutation, but also somatic mutation can lead to development of a tumor associated with the VHL disease.

The effect of iodine-based contrast agents on the levels of radiation-induced chromosomal aberrations

The effects of iodine-based contrast agents on the repair of radiation-induced chromosomal damage were investigated employing peripheral blood from a healthy male donor. The blood samples were irradiated with 0.5-4.0 Gy 137Cs gamma rays. Contrast agents and NaCl solutions of various concentrations were added to the blood within the first 15 min or at 60 min after irradiation, and the samples were subsequently cultured for 45 h at 37 degrees C. Significantly elevated frequencies of chromosomal abnormalities caused by postirradiation treatment with hypertonic contrast agents appeared to increase with increasing hypertonicity. Elevated aberration frequencies were found to be greatest in the samples treated within 15 min of irradiation. The contrast agents had little effect if they were added at 60 min after irradiation, probably because the process of chromosome rejoining had been completed. Isotonic iodine-based contrast agents did not enhance the frequencies of chromosomal aberrations to a significant degree.

DNA fragmentation induced by a cytoplasmic extract from irradiated cells

Apoptosis is a mode of cell death characterized by distinct morphological features and DNA fragmentation. The program that leads to apoptosis has been considered to be predominantly extranuclear, and a signal transduction pathway to the nucleus exists during apoptosis, while characteristic events occur in the nucleus. As for radiation-induced apoptosis, the signal transduction pathway remains unclear, especially the sites where the primary effect of radiation occurs. In this study, we demonstrate that a cytoplasmic extract prepared from irradiated cells has the ability to cause DNA fragmentation and that caspase-3 is activated in this extract. Normal nuclei of HeLa S3 cells were added to a cytoplasmic extract made from HL60 cells which had been irradiated with 30 Gy of 137Cs gamma rays and were incubated. Agarose gel electrophoresis of the added nuclei showed a characteristic DNA laddering pattern. This reaction was blocked by a caspase-3 inhibitor but not by an ICE inhibitor. These observations suggest that a signal transduction pathway from an unknown target of gamma radiation may exist upstream of caspase-3 during radiation-induced apoptosis.

Comparison between Fractionated High Dose Rate Irradiation and Continuous Low Dose Rate Irradiation in Spheroids

Recent interest in clinical brachytherapy focuses on the possible radiobiological equivalence between fractionated high dose rate (HDR) and continuous low dose rate (LDR) irradiations. This study is designed to compare the radiobiological effects between the two in vitro using multicellular spheroids of human tumor. Both HDR and LDR irradiations were delivered by 137Cs source, the dose rates of which were as 1.18 Gy/min and 5.5 mGy/min, respectively. Fractionated HDR irradiation of various fraction sizes was applied twice a day. We found that: (1) The fractionated HDR irradiation (8 Gy/2 fr/day) was more effective radiobiologically than continuous LDR irradiation (8 Gy/day) and the ratio of radiobiological effects of these irradiations was estimated as 0.82, based on the 50% spheroid cure dose (SCD50); (2) the radiobiological effectiveness was independent of the fraction size of HDR irradiation administrated, and the repair of sublethal damage (SLD) was absent, suggesting that the sparing effect of fractionated HDR irradiations was absent in spheroids. Our findings could provide important information for the clinical usage of the fractionated HDR radiotherapy to replace continuous LDR radiotherapy.