Effects of 5-fluorouracil on cell cycle arrest and toxicity induced by X-irradiation in normal mammalian cells.

UNLABELLED: From clinical studies in cancer patients and experimental in vitro studies, there is evidence of an increased cytotoxic effect, and even synergy, when irradiation is combined with 5-fluorouracil (5-FU). The mechanism for this is unclear. MATERIALS AND METHODS: Mouse fetuses (C3H) have been exposed in vivo to X-irradiation and 5-fluorouracil (5-FU) as single agents or in combination. Cell proliferation, cell cycle progression, fetal survival and incidence of fetal malformations have been studied. PURPOSE: The aim of this study was to determine possible synergistic cytotoxic effects when 5-FU and ionizing radiation were combined, particularly concerning the regulation of cell cycle progression in proliferating, non malignant mammalian cells in vivo. RESULTS: The combination of low-toxic doses of X-irradiation and 5-FU had a synergistic toxic effect in nonmalignant mouse fetuses in vivo. The cell cycle regulation was perturbed and the radiation-induced G2-arrest was eradicated by 5-FU during the initial hours. CONCLUSIONS: The time for repair of radiation induced DNA-damage is probably reduced, which may explain the increased toxicity of this combination.

Cisplatin-induced inhibition of p34cdc2 is abolished by 5-fluorouracil.

Clinical (Dimery and Hong, J Nat Cancer Inst 1993; 85: 95- 111) and experimental studies (Scanlon et al., Proc Natl Acad Sci USA 1986; 83: 8923-5; Lewin et al., In Vivo 1990; 4: 277-82) have indicated an increased cytotoxic effect, when cisplatin (CDDP) is combined with 5-fluorouracil (5-FU). Addition of 5-FU abolishes the G2-arrest induced by CDDP (Lewin et al., In Vivo 1990; 4: 277-82; Nylén et al., Acta Oncol 1996; 35: 229 35). The mechanism for the synergy is unclear. Activation of p34cdc2 is necessary for progression from G2 to mitosis (Lewin et al., Anti-Cancer Drugs 1995; 6: 465-70). The aim was to study p34cdc2, cdc25C and weel after treatment of mammalian tumour cells in vivo with CDDP as single agent or in combination with 5-FU. CDDP prevented activation of p34cdc2 by keeping cdc25C inactive and weel active. Addition of 5-FU to CDDP decreased the expression of weel and promoted cdc25C-activation. p34cdc2 was dephosphorylated by cdc25C and activated. Alterations in activity of cdc25C and weel after drug combination were due to changes in the protein amount, rather than to changes in the phosphorylation degree.

Absence of misincorporation of pyrimidines in DNA after treatment with a combination of cisplatin (CIS-diammine-dichloro-platinum) amd 5-fluorouracil of mouse sarcoma cells.

The effects on incorporation into DNA of the deoxyribonucleotides dCTP and dTTP and the DNA synthesis rate after treatment with cisplatin (CDDP), 5-fluorouracil (5-FU) or a combination of CDDP and 5-FU were studied in ascites sarcoma (Bp8) growing in mice. Single administration of CDDP gave an early (1 h) transient increase in the DNA-synthesis followed by a decrease. 5-FU as single agent did increase the rate of DNA synthesis after 6 h with a maximum at 10 h. The combination of CDDP and 5-FU markedly increased the rate of DNA synthesis up to 6 h as compared to single drug treatment. Although the dCTP pool increased after combined treatment, while the dTTP pool was unchanged, no alterations in the proportions of dTTP and dCTP incorporated into DNA could be detected. Hence, misincorporation of pyrimidines is not the mechanism for the synergistic effect of the combination of CDDP and 5-FU.

Standardized precision radiotherapy in choroidal metastases.

Metastases in the choroid of the eye are frequent in patients with disseminated malignancy. We here report the results using the precision radiotherapy technique described by Schipper et al. to treat 14 of 17 consecutive patients (21 eyes) with symptoms from such metastases. A beam defining collimator was used and a lateral field was given with the treated eye individually fixed. Varying fractionations and doses were used. The biologically effective dose for early effects (BED3) was 47 to 90 Gy and for late effects (BED10) 28 to 59 Gy. In 14 eyes (82%) the metastases regressed completely. The visual acuity was stabilized or improved in all patients and none needed local surgery. Three patients developed signs of radiation retinopathy, but only in one case the visual function was compromised. With this standardized technique no individualized dose planning was needed, the risk of radiation cataract was minimized and a dry eye avoided.

Gene amplification detected in carcinoma cells from human urinary bladder washings by the polymerase chain reaction method.

BACKGROUND: Urinary bladder carcinoma often is diagnosed from malignant cells in bladder washings obtained at cystoscopic examination. In some cases, there are difficulties distinguishing between cytologic Grades 1 and 2. Detection of genetic alterations in combination with morphologic analysis may facilitate the diagnosis. METHODS: The presence of amplified c-erb-B2 and epidermal growth factor receptor (EGF-R) was examined in tumor cells present in bladder washings. The gene copy number was determined with the polymerase chain reaction (PCR). RESULTS: The authors detected amplified c-erb-B2 and EGF-R in cancer cells of cytologic Grade 2 and 3 tumors. They did not detect amplification in cytologic Grade 1 tumor cells or in cells from bladders, without known malignant neoplasms. CONCLUSIONS: Examination of genetic alterations, in combination with morphologic analysis, facilitates the diagnosis of carcinoma.

Appearance and detection of multiple copies of the mdr-1 gene in clinical samples of mammary carcinoma.

To examine the presence of multiple copies of the mdr-I gene in clinical tumor samples we have developed an approach where the cells are obtained by fine-needle biopsies and the number of gene copies determined by PCR. The temporal appearance of amplified mdr-I was examined in 20 breast-cancer patients with clinical stage-IV disease receiving endocrine treatment. Tumor samples were obtained every 2nd to 3rd month from the same tumor lesion. None of the initial samples from each patient contained multiple copies of mdr-I. Of 16 patients who showed increased tumor size, 4 developed multiple gene copies, showing that the event occurs without cytotoxic selection of cells with chemotherapy.

Detection and temporal appearance of multiple copies of c-erb-B2 genes in advanced mammary carcinoma using fine needle biopsies and the polymerase chain reaction.

Aspiration of tumor cells by the fine-needle biopsy method yields only a small number of cells, which hampers conventional molecular analysis for the presence of multiple copies of oncogenes. We have therefore adopted the polymerase chain reaction (PCR) method to study semi-quantitatively the level of the c-erb-B2 gene in human breast tumor samples. Of 39 patients with mammary carcinoma, 7 (19%) contained multiple copies of c-erb-B2 genes, whereas only two samples failed to give informative data. Next the multiple copies of c-erb-B2 genes, whereas only two samples failed to give informative data. Next the temporal appearance of multiple gene copies was examined in 20 patients with clinical stage IV disease. Tumor samples were obtained every second to third month from the same tumor lesion of each patient. None of the initial samples from each patient contained multiple copies of c-erb-B2. Of 16 patients that showed progressive clinical disease, 5 developed multiple gene copies, showing that the event occurs in clinical stage IV disease.

Amplification of oncogenes in mammary carcinoma shown by fine-needle biopsy.

A procedure that measures the amplification of oncogenes in human cancer cells is described. The cells were obtained by fine-needle biopsy to allow repeated sampling from individual metastases. A drawback was the low number of cells obtained, but this could be overcome by using a slot-blot hybridization technique to measure gene amplification. Two patients with mammary cancer (primary tumors or metastases), analyzed for the levels of amplification of the oncogene erb-B2, are described in detail. This technique is suitable for analyzing alterations occurring during cancer progression and for identifying subgroups of mammary cancer with different characteristics.

An abnormal profile of DNA replication intermediates in Bloom's syndrome.

Bloom's syndrome (BS) cells display a characteristic genomic instability, notably an elevated frequency of sister-chromatid exchange. Replicating DNA in cultured BS cells was labeled with [3H]thymidine using several time schedules. Separation of DNA in agarose gels showed high molecular weight DNA and three classes of DNA replication intermediates: 20-kilobase DNA, 10-kilobase DNA, and Okazaki fragments. In contrast newly replicated DNA from normal cells showed no 20-kilobase DNA replication intermediates. Certain BS cells, exceptional in that their characteristic genomic instability has for unknown reasons been corrected, also differed from normal cells in having the 20-kilobase intermediate, but they differed from both normal cells and the other (the uncorrected) BS cells in lacking the 10-kilobase DNA replication intermediates.

Increased levels of 5-fluorouracil-induced DNA lesions in Bloom's syndrome.

In Bloom's syndrome (BS) the regulation of uracil-DNA glycosylase, an enzyme involved in the repair of DNA containing 5-FU, is altered. 5-FU induces higher levels of DNA fragmentation in BS cells than in non-BS cells. The increase in DNA fragmentation is connected to the cytotoxic mechanism where 5-FU is incorporated into DNA. When 5-FU induces DNA fragmentation by a mechanism not involving the incorporation of drug into DNA, the levels of DNA fragmentation in BS and non-BS cells remain similar.

Bleomycin-induced DNA lesions are dependent on nucleosome repeat length.

Treatment of cells or nuclei with bleomycin induces DNA lesions. We detect the presence of lesions as the release of fragments from bulk DNA when cells (or nuclei) are lysed in dilute alkali. To further characterize the lesions we have altered experimentally the average nucleosome repeat length and probed the lysate with nuclease S1 in order to remove single-stranded DNA. In salt-incubated nuclei with short average nucleosome repeat length (140-145 base pairs) (and also with long nucleosome-free stretches of DNA) one can induced fewer DNA lesions in the nucleosome-containing DNA as compared to nuclei with 190-195 base pairs average nucleosome repeat length. Hence the ability of bleomycin to induce DNA lesions is dependent on nucleosome repeat length.

Altered formation of DNA in human cells treated with inhibitors of DNA topoisomerase II (etoposide and teniposide).

We have investigated the importance of DNA topoisomerase II for the formation of mammalian DNA replication intermediates. Treatment with the DNA topoisomerase II inhibitor etoposide (teniposide) prevents the formation of large intermediates, such as 10-kilobase DNA, but allows the formation of small intermediates, i.e., Okazaki fragments. In untreated cells, there is a distinct stage in which the 10-kilobase DNA intermediates are joined before the appearance of mature chromatin. We find that pretreatment with etoposide (teniposide) prevents the appearance of this stage. When the protocol is reversed and the cells contain labeled 10-kilobase DNA before exposure to the drugs, one can detect the stage.

Appearance of a late stage during mammalian DNA replication when cells resume formation of 10 kb DNA replication intermediates.

After the joining of human large DNA replication intermediates and before the appearance of mature chromatin DNA, there exists a distinct stage--'the post-elongation stage'. This stage reappears during recovery of DNA synthesis simultaneously with the reappearance of a large DNA replication intermediate, 10 kb DNA.

Altered formation of DNA replication intermediates in human 46 BR fibroblast cells hypersensitive to 3-aminobenzamide.

The appearance of DNA replication intermediates was investigated in a human fibroblast strain (46 BR) which is hypersensitive to the lethal effects of 3-aminobenzamide. 3-Aminobenzamide is an inhibitor of poly(ADP-ribose) synthetase and modulates DNA ligase activity. We detected the same intermediates (10 kb DNA and Okazaki-fragments) as in normal fibroblasts, but kinetics and amounts of intermediates were altered, either as a result of, or in order to overcome the defect in the cells.

Reduced DNA damage induced in human melanoma cells by dacarbazine in the presence of deoxyribonucleosides.

Decarbazine induces DNA lesions in replicating DNA. We show here that deoxyribonucleosides, with the exception of thymidine, in doses of 250 microM or higher, prevent dacarbazine-induced DNA lesions. The DNA lesions that appear in the presence of thymidine can be prevented by aphidicolin, an inhibitor of DNA synthesis. Cytotoxicity analyses confirm that the combination thymidine-dacarbazine prevents cell outgrowth only partially, while other deoxyribonucleosides completely abolish the dacarbazine effect.

5-Fluoropyrimidine-induced DNA damage in human colon adenocarcinoma and its augmentation by the nucleoside transport inhibitor dipyridamole.

5-Fluorouracil and 5-fluorodeoxyuridine induce DNA lesions via two different mechanisms, one involving and the other not involving the incorporation of drug into DNA. The DNA lesions are detected by lysing cells in dilute alkali and then separating the DNA in agarose gel electrophoresis. We examine here the effect of dipyridamole, a nucleoside transport inhibitor, on the DNA lesions. We find that dipyridamole augments the levels of DNA fragmentation when the lesions are induced by the mechanism not involving the incorporation of drug. In parallel cytotoxicity is increased.

Weekly doxorubicin in the treatment of metastatic breast carcinoma.

The introduction of aggressive combination chemotherapy has not been accompanied by any significant prolongation of survival in metastatic breast carcinoma. Hence, there is a need of less toxic but--in terms of palliation--effective chemotherapeutic regimens. This paper concerns 50 patients with progressive metastatic breast carcinoma who were treated with weekly bolus injections of doxorubicin (15-20 mg). All patients were followed until disease progression. A partial response with a mean duration of 6 months was achieved in 7 patients (14%). In addition, stabilization of disease was observed in 24 patients (48%) during 2-34 months. The toxicity was generally mild. It is concluded that weekly doxorubicin with fairly low doses is a moderately effective treatment in stage IV breast carcinoma. It is devoid of severe toxicity which makes it useful even in old and debilitated patients.

Altered formation of DNA replication intermediates in cells growing in different culture conditions.

In human melanoma cells one can detect two discrete DNA replication intermediates, 10-kb DNA intermediates and Okakzaki fragments. Both intermediates are seen when cells are rapidly growing in medium supplemented with fetal calf serum. When the medium is supplemented with newborn calf serum, one can detect Okakzaki fragments but not 10-kb DNA intermediates. In contrast we do not detect changes in the replicon sizes in the two media.