Taketoshi Sugiyama

Factors involved in differential Giemsa-staining of sister chromatids.

Microspectrophotometric evaluation of differentially stained sister chromatids made it possible to analyse precisely the factors involved in the Giemsa methods. The concentration of Hoechst 33258, pH of the mounting medium, temperature during UV-exposure and the quality (wavelength) of UV-light influenced the differential staining. Exposure of blacklight of 10−5 M Hoechst 33528-stained BrdU-labeled chromosome specimens mounted in McIlvaine buffer (pH 8.0) at 50° C reproducibly allowed differential staining of sister chromatids within 15 min. On the other hand, Korenberg-Freedlenders method using no Hoechst 33258 was also UV-light-dependent. Thus, photolysis of BrdU-substituted DNA was considered the basic mechanism of the Giemsa methods where the photosensitive Hoechst 33258 played a role as a sensitizer.

Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining

The essential steps of the 33258 Hoechst-Giemsa method for differential chromatid staining consist of (1) 33258 Hoechst treatment, (2) exposure to light, and (3) Giemsa staining. The staining was shown to be a function of the concentration of 33258 Hoechst and the light exposure. The dye was successfully replaced by various metachromatic dyes such as thionine. Two simple methods are proposed. Failure of the pale stained chromatids to restore Giemsa affinity with urea and trypsin and the diminished Feulgen reaction after light exposure suggest that not masking proteins but photolysis of the BrdU-incorporated chromatid components in the presence of photosensitive dyes play a role in the differential staining.

A summary of cytogenetic studies on 534 cases of chronic myelocytic leukemia in Japan

Cytogenetic and clinical data on 534 patients with chronic myelocytic leukemia (CML) were collected from 10 institutions in Japan. The results of the analysis of the data were in substantial accord with those of the First International Workshop on Chromosomes in Leukemia and other published data, but certain differences were noted in the frequency of Philadelphia chromosome (Ph1)-negative cases, unusual and complex Ph1 translocations, and additional chromosome changes. Some of the findings are discussed with respect to the origin of unusual and complex Ph1 translocations, the relationship between chromosome abnormalities and survival, and geographic differences in chromosome abnormalities.

The location of ribosomal cistrons (rDNA) in chromosomes of the rat

In situ hybridization of 3H-labelled ribosomal RNA to the chromosomes of rat bone marrow cells revealed that clusters of ribosomal cistrons (rDNA) are located in the secondary constrictions of chromosomes No. 3 and 12 and near the centromere of chromosome No. 11, both associated with the late DNA-replicating regions. They were not found in Nos. 1, 2, 13, 19, 20, and the Y chromosome.

Suppression of 7,12-dimethylbenz[a]anthracene-induced chromosome aberrations in rat bone marrow cells by vegetable juices

A study was made of the in vivo effects of various vegetable juices on 7,12-dimethylbenz[a]anthracene (DMBA)-induced chromosome aberrations (CA) in rat bone marrow cells. DMBA-induced CA consisted mainly of gaps and breaks. Exchanges were observed infrequently. Depending on the progressive severity of their chromosome damage, cells were classified into 4 categories: cells with gaps only, cells with breaks, cells with exchanges, and cells with multiple CA (more than 10 aberrations). Multiple Ca and the number of aberrations per cell, reflecting the severity of damage within a cell, were significantly suppressed by most vegetable juices investigated. The effect of fresh or boiled juices from 10 vegetables on the incidence of DMBA-induced aberrant metaphase cells (not including cells with gaps) revealed significant suppression of the incidence by fresh or boiled juice from onion, burdock, egg plant, cabbage and welsh onion. There was no difference between the effect of fresh juice or boiled juice except in the pumpkin. Fresh pumpkin juice, conversely, enhanced the incidence of aberrant cells, while boiled pumpkin juice significantly suppressed it. The present results may suggest that some vegetables, such as onion, suppress chemically induced cancer. Glutathione also suppressed DMBA-induced CA; it is, of course, well known that SH compounds analogous to GSH are plentiful in onion and welsh onion.

Chromosome Abnormality in Rat Leukemia Induced by 7,12-Dimethylbenz[a]anthracene

A high percentage of consistent chromosome abnormality, trisomy of the longest telocentric chromosome, was found in leukemias induced in rats of the Long-Evans strain by pulse doses of 7,12-dimethylbenz[a]anthracene. Cells with this abnormality were large, immature, and mononuclear and tended toward erythroblastic maturation.

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

SummaryAn in vivo 5′-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%–20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.

Mixed connective tissue disease with fatal pulmonary hypertension and a review of literature

The paper presents an autopsy case of mixed connective tissue disease (MCTD) with pulmonary hypertension (PH) and a review of literature. A 33-year-old woman with Raynauds phenomenon and dyspnea of one year duration was diagnosed as having MCTD on the basis of a higher titer (1:163,840) of serum antibodies to the ribonucleoprotein (RNP). Cardiac catheterization showed complicating PH, confirmed an autopsy by the findings of concentric intimal cellular proliferation and typical plexiform lesions in the small arteries and arterioles of the lung, suggesting primary PH. Fatal PH with MCTD has been reported only 6 cases in literature including our case. All were young females, with histopathological findings consistent with plexogenic pulmonary arteriopathy in 5 cases and with recurrent pulmonary thromboembolism in the other. The aetiology of PH is still unknown, but it may be due to vasoconstriction evoked by the hyper-reactivity of the vessels.

Acute cytogenetic effect of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2, a food preservative) on rat bone marrow cells in vivo

The cytogenetic effects of 2(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2), a food preservative used in Japan, on rat bone marrow cells in vivo were studied. The aberrant metaphase cells in the bone marrow increased and reached the peak level 6 h after intraperitoneal injection of 240 mg/kg body weight of AF-2 and returned to the normal level within 24 h. A dose-response relationship was obtained using 4-240 mg/kg of AF-2. Chromosome aberrations were also induced after oral administration of 30-240 mg/kg. The aberrations were mostly chromatid breaks and the distribution among and within chromosomes was similar to those induced by a carcinogen, 7, 12-dimethylbenz(a)anthracene (DMBA). The present results provide the first evidence of in vivo cytogenetic effects of AF-2 on mammalian cells and, together with the evidence of mutagenicity already proved in other organisms, warn of the possible genetic hazards to cells exposed to this compounds.

Enhanced Inhibition of Anticancer Drugs by Human Recombinant Gamma-Interferon for Human Renal Cell Carcinoma in Vitro

We studied the inhibitory effect of seven anticancer drugs and alpha-, recombinant beta-, and recombinant gamma-interferons on the in vitro growth of two established human renal cell carcinomas and 16 renal cell carcinomas obtained from patients using monolayer culture and the double-layer soft agar system. Recombinant gamma-interferon was the most effective of three types of interferon. Combined treatment with recombinant gamma-interferon and some anticancer drugs inhibited the cell growth in both cell lines more than treatment with each drug alone. Treatment with recombinant gamma-interferon and cisplatinum or 5-fluorouracil following a 24-hour incubation with the interferon was more effective than when interferon and the drug were given simultaneously. Treatment using doxorubicin, cisplatinum, or vinblastine with recombinant gamma-interferon synergistically inhibited colony formation in 11 of the 16 renal cell carcinomas that showed clonal growth. Our results suggest that treatment with anticancer drugs in combination with recombinant gamma-interferon is effective for renal cell carcinoma.