Detection of APC mutations by a yeast-based protein truncation test (YPTT).

APC gene mutations play a role in the initiation step of colorectal carcinogenesis in both familial adenomatous polyposis (FAP) and non-FAP patients. Almost all of the APC mutations are nonsense or frameshift mutations, which truncate the APC protein and are thought to inactivate normal APC function. We show a novel method for detecting nonsense and frameshift APC gene mutations by using Saccharomyces cerevisiae. Polymerase chain reaction (PCR)-amplified APC fragments are cloned directly into yeast expression vectors in vivo, and the yeast expresses a hemagglutinin epitope (HA)-tagged APC peptide. When an APC fragment contains a nonsense or frameshift mutation, HA-tagged truncating APC peptide can be detected by Western blotting using an anti-HA antibody. We identified both germ-line and somatic APC mutations in patients with FAP and non-FAP colorectal tumors, respectively. This method, called the yeast-based protein truncation test (YPTT), is simple and fairly cheap, and it can be applied to any genes that are inactivated by protein truncating mutations.

Undifferentiated sarcoma of the liver in a 21-year-old woman: case report.

A successful surgical case of malignant undifferentiated (embryonal) sarcoma of the liver (USL), a rare tumor normally found in children, is reported. The patient was a 21-year-old woman, complaining of epigastric pain and abdominal fullness. Chemical analyses of the blood and urine and complete blood counts revealed no significant changes, and serum alpha-fetoprotein levels were within normal limits. A physical examination demonstrated a film, slightly tender lesion at the liver's edge palpable 10 cm below the xiphoid process. CT scan and ultrasonography showed an oval mass, confined to the left lobe of the liver, which proved to be hypovascular on angiography. At laparotomy, a large, 18 x 15 x 13 cm tumor, found in the left hepatic lobe was resected. The lesion was dark red in color, encapsulated, smooth surfaced and of an elastic firm consistency. No metastasis was apparent. Histological examination resulted in a diagnosis of undifferentiated sarcoma of the liver. Three courses of adjuvant chemotherapy, including adriamycin, cis-diaminodichloroplatinum, vincristine and dacarbazine were administered following the surgery with no serious adverse effects. The patient remains well with no evidence of recurrence 12 months after her operation.

[Subrenal capsule assay using nude mice].

Six-day subrenal capsule assay (SRCA) using normal immunocompetent mice developed by Bogden et al. is a promising in vivo chemosensitivity test. This method, however, has a problem of influence of the host reaction. We compared the tumor growth kinetics and host reaction between normal immunocompetent and nude mice. The tumor diameter increased until day 6 in normal and day 16 in nude mice, respectively. However the histological finding revealed many host reactive cells and few viable tumor cells on day 6 in normal mice, and well preserved tumour cells on day 16 in nude mice. These results were supported by flow cytometrical analysis. Then, we examined two immunosuppressive drugs; cyclophosphamide (EX) and cyclosporin A (CSA) in SRCA. The tumors increased in the diameter until day 16 in both EX and CSA-treated groups, but the results of the histological examination showed that tumor cells were preserved in tissue on day 14 in CSA-treated and day 6 in EX-treated group. These results were also supported by flow cytometrical analysis. From the investigation of antitumour activities of adriamycin (ADR) and mitomycin C, it was suggested that the 12-day assay was suitable if nude mice were used in SRCA, and six-day assay also, if EX-treated normal mice were used. In CSA-treated group, more toxicity of anticancer drugs was manifested than usual. We studied whether or not CSA had a usefulness in SRCA with normal immunocompetent mice. Sixty mg/kg of CSA was given to BDF1 mice daily SC, and various dosage of ADR was given i.v. on day 2. The body weight of BDF1 mice decreased over 20% within 10 days when ADR was given at more than 5 mg/kg. MX-1, a human breast carcinoma cell line, is known to be sensitive to ADR. This tumor was implanted SC in the back of BALB/c nu/nu mice and chemosensitivity was tested against ADR. ADR resulted to be positive at the dose of 8 mg/kg. On the other hand, the dose of 5 mg/kg proved to be negative, and hence the result of SRCA would be false negative, if the dose of ADR is reduced to avoid the toxicity of CSA. The tumor grew slowly when only 60 mg/kg of CSA was given daily for three weeks, and the inhibition rate was 56.2%. The toxicity of CSA was neglected because the body weight loss was approximately 13%. CSA may have the antitumor effect by itself, and EX did not suppress the host reaction sufficiently.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of mizoribine as an immunosuppressant in subrenal capsule assay using immunocompetent mice.

We studied the application of mizoribine (MZR) to normal immunocompetent mice in subrenal capsule assay (SRCA) by means of tumor growth curve determination, histological analysis and autoradiography. At 400 mg/kg, MZR prolonged the actual tumor growth and moderately reduced the host reaction. Doses below 200 mg/kg did not effectively suppress the host reaction. The maximal weight loss of mice in the 400 mg/kg group reached 29%, but did not exceed 10% within 8 days. Hence, we applied 400 mg/kg of MZR to SRCA for up to eight days for cancer chemotherapy testing. This dose of MZR did not affect the labeling index of tumor cells compared with the control.

[In vivo antitumor activity of mitoxantrone and the flow cytometric analysis of its influence on cell cycle transition--comparison with doxorubicin and aclarubicin on ascitic hepatoma AH109A cells].

Mitoxantrone was compared with doxorubicin and aclarubicin of its in vivo antitumor activity and influence on cell cycle transition by use of rat ascitic hepatoma AH109A. Antitumor activity determined by the cell growth curve was similar in mitoxantrone and doxorubicin, but the sensitivity of AH109A to aclarubicin was lower than that to the other two drugs. Doxorubicin and mitoxantrone showed all phase arrests with 1/10 of maximally tolerated dose (MTD), and with lower concentrations a strong arrest at G2 phase was observed, thus, mitoxantrone appeared to have a similar antitumor activity on AH109A to that of doxorubicin. Aclarubicin, with 1/10 MTD, demonstrated only a transient arrest at G2 phase, cells arrested at G2 phase entering into the next phase. With below 1/10 MTD, there was no appearance on histograms, and the influence on AH109A cell cycle transition by aclarubicin was considered to be little in comparison with doxorubicin and mitoxantrone.

[Long-term arterial infusion chemotherapy in unresectable pancreatic cancer].

Four patients with unresectable pancreatic cancer and one with metastatic liver tumor after curative pancreatic resection for carcinoma of the pancreas had undergone intra-arterial infusion chemotherapy using an implantable drug delivery system. Concentration of the anti-cancer drug (CDDP) was measured in the specimen of cancer tissue obtained following intra-arterial infusion of the drug with angiotensin II. The concentration of the drug was 1.3 times higher in the cancer tissue than in the adjacent normal tissue in two of three patients. No remarkable response was observed in terms of tumor size and survival time. However, the value of CA 19-9 was reduced considerably after chemotherapy in two cases. Intra-arterial chemotherapy using a drug delivery system can be carried out safely and easily even in the outpatient department and can contribute to the quality of life and prognosis of patients.

[An experimental study on subrenal capsule assay (SRCA)--problems for the use of immunosuppressive agents].

We studied whether or not cyclosporin A (CSA) has a usefulness in subrenal capsule assay (SRCA) with normal immunocompetent mice. Sixty mg/kg of CSA was given to BDF1 mice daily subcutaneously, and various dosages of adriamycin (ADR) was given intravenously on day 2. The body weight of BDF1 mice decreased over 20% within ten days when ADR was given at more than 5 mg/kg. MX-1, a human breast carcinoma line is known to be sensitive to ADR. This tumor was implanted subcutaneously in the back of BALB/c nu/nu mice and chemosensitivity was tested against ADR. ADR resulted to be positive at the dose of 8 mg/kg. On the contrary, the dose of 5 mg/kg proved to be negative, and hence the result of SRCA would be false negative, if the dose of ADR is reduced to avoid the toxicity of CSA. The tumor grew slowly when only 60 mg/kg of CSA was given daily for three weeks, and the inhibition rate was 56.2%. The toxicity of CSA was neglected because of the body weight loss was approximately 13%. CSA may have the antitumor effect by itself, and we therefore suggest that the CSA is not useful for SRCA.

[An experimental study on SRCA (subrenal capsule assay)--using immunosuppressive maneuvers].

Six-day SRCA using normal mice developed by Bogden et al. is one of the most promising methods for in vivo chemosensitivity tests. However, this method has a problem on the influence of the host reaction during six days. Therefore, we examined the tumour growth kinetics, host reaction and expression of antitumor effect on three immunosuppressive maneuvers; cyclophosphamide (EX), cyclosporin A (CSA), and total body irradiation (TBI). The tumour diameter increased until day 16 in EX and CSA-treated groups and day 10 in TBI-treated group, but the results of the histological examination showed that tumour cells were preserved in tissue on day 14 in CSA-treated group and day 6 in EX and TBI-treated groups. These results were supported by flow cytometrical analysis. The autoradiogram using 3H-TdR showed that labelling index of the tumour cells was not affected by these immunosuppressive maneuvers. From the investigation of the antitumour activity of adriamycin and mitomycin C, it was suggested that the 12-day assay was suitable if nude mice were used in SRCA, and six-day assay, if EX-treated normal mice were used. In CSA-treated group, toxicity of anticancer drugs was manifested than usual.

Inhibitory effects of prostaglandin A2 on c-myc expression and cell cycle progression in human leukemia cell line HL-60.

The effect of prostaglandin A2 (PGA2) on c-myc expression was investigated in a human promyelocytic leukemia cell line, HL-60, which responded to PGA2 with a dose-dependent growth inhibition. Northern blot analysis indicated that treatment with PGA2 at 0.5 to 5.0 micrograms/ml remarkably reduced the steady state level of c-myc mRNA within 3 h, and then it gradually recovered according to the order of concentration of the drug. In contrast to c-myc, the level of class I HLA mRNA, as an internal control, was not diminished by PGA2 treatment. Further, this reduction of c-myc was not disturbed by cycloheximide, suggesting that this PGA2 action on c-myc expression is independent of de novo protein synthesis. Cytofluorometric analysis revealed that the exposure of HL-60 cells to PGA2 at 0.5 or 5.0 micrograms/ml arrested the cells in the G0-G1 phase of the cell cycle. This accumulation of the cells in G0-G1 phase continues until 24 or 36 h at 0.5 or 5.0 micrograms/ml, respectively. The G0-G1 arrest of the cell cycle was also recovered as the inhibition of c-myc was released. This recovery may be due to the loss of activity of PGA2 in culture medium. This study clearly showed that PGA2 treatment arrested HL-60 cells in the G0-G1 phase of the cell cycle and was associated with the reduction of c-myc mRNA.

[An experimental study on SRCA (subrenal capsule assay)--comparison of the test between normal and nude mice].

Six-day SRCA using normal mice developed by Bogden et al. is a promising in vivo chemosensitivity test. However, this method has a problem on the influence of the host reaction. We compared the tumor growth kinetics and host reaction between normal and nude mice. The tumor diameter increased until day 6 in normal and day 16 in nude mice, but the histological finding revealed many host reactive cells and few viable tumour cells on day 6 in normal mice, and well preserved tumour cells on day 16 in nude mice. These results were supported by flow cytometrical analysis. Autoradiogram using 3H-TdR showed a recovery of labeling index to the steady label by day 1. This index was similar between normal and nude mice. When antitumor activity of adriamycin, cisplatin or mitomycin C was compared with nude mice system, the order of effectiveness was the same as the system using nude mice implanted tumor cells subcutaneously and given the drugs intraperitoneally, but different in normal mice.

The mechanism of action of quinocarmycin citrate (KW 2152) on mouse L1210 cells in vitro.

The effects of the antitumor antibiotic, quinocarmycin citrate (KW 2152), on L1210 cells were studied in vitro. The cellular growth was completely inhibited at 10(-6) M KW 2152, and after 2 days no viable cell was seen. The incorporation of 3H-thymidine, 3H-uridine, or 3H-leucine into the acid-insoluble fraction was not affected at 10(-4) M for 1 h; however, when the cells were treated with 10(-6) M for 24 h, the radioactivity appearing in the acid-insoluble fraction was reduced to 20%, 30%, and 48%, respectively, of the control. The single strand scission of the DNA of L1210 cells was seen at 10(-7) M for 24 h, as revealed by an alkaline, sucrose density gradient. However, no damage to plasmid pBR322 was observed even at 10(-6) M KW 2152 for 24 h, as revealed by 0.8% agarose gel electrophoresis, indicating that some soluble factors of the cells might contribute to the damage to the DNA of L1210 cells. The processing of pre-rRNA of the cells was not inhibited at 10(-6) M of the drug for 24 h of incubation.

Studies on the mechanism of action of ACNU, 1-(4-amino-2-methylpyrimidine-5-yl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride: effects on cultured HeLa S3 cells.

Treatment of cultured HeLa S3 cells with ACNU, 100 microgram/ml, for 30 min inhibited the cell growth intensely. The cell number was minimum on the 4th day after the treatment and recovered gradually, but it was still 24% of control of the 14th day. The colony formation, as an index of proliferation ability of cells, was remarkably inhibited and the number of colony formed on the 14th day after the treatment was 8% of control. DNA synthesis was inhibited by 59%, whereas little or no effect was observed on RNA or protein synthesis after 24 hr. Ethylene-14C-ACNU was bound to DNA and RNA to a similar degree, and that bound to the acid-insoluble fraction within 30 min was decreased by half after 24 hr and sustained thereafter. A significant decrease in sedimentation velocity of DNA on alkaline sucrose density gradient centrifugation was observed after 24 hr, and the decrease on neutral sucrose density gradient centrifugation was noticeable already by 2 hr. From the results of this study and other reports, the mechanism of action of ACNU seems to be alkalization of DNA followed by damage to DNA, which progresses quite slowly compared with other alkylating agents, causing cell damage and cell death.

Damage of DNA and its recovery in AH-109A cells treated with carboquone in vivo.

A single injection of carboquone at a dose of 0.1 mg/kg body weight induced damage of DNA of AH-109A cells as revealed by alkaline and neutral sucrose density gradient centrifugation. A significant decrease in the sedimentation velocity of DNA on alkaline sucrose density gradient centrifugation was observed 15 min after the injection, and it returned to that of control after 60 min. Thereafter, the size of DNA decreased progressively. When the cells were lysed with 2% sodium dodesyl sulfate solution and analyzed on neutral sucrose density gradient centrifugation, a similar change in the sedimentation velocity was observed. The results obtained from the studies of intraperitoneal growth of AH-109A cells pretreated with carboquone in vivo and the survival of host animals well corresponded to the extent of the damage of DNA as revealed by alkaline and neutral sucrose density gradient centrifugation.