Toxicity, pharmacokinetics and pharmacodynamics of the Soviet bleomycin, bleomycetin, in a single administration to laboratory animals]

Toxicity of bleomycetin (bleomycin A2) administered intravenously, intraperitoneally, subcutaneously or intramusculary in a single dose to animals was almost identical. On its oral administration bleomycetin was 10--14 times less toxic than on its parenteral use. Rats were somewhat less sensitive to bleomycetin than mice. Bleomycetin had no significant effect on the level of the arterial pressure, respiration, ECG characteristics and elements of the vegetative nervous system in narcotized cats. After a single intravenous or subcutaneous administration to rabbits bleomycetin was detectable in the blood for 4--5 hours. The highest bleomycetin levels were registered in the skin, kidneys and lungs. Bleomycetin was mainly excreted with the urine.     

Use of bleomycetin in malignant testicular tumors and the problem of studying bleomycin analogs

The efficacy of the chemotherapy of 16 patients with metastases of malignant tumors of the testicle was studied. The chemotherapy included vinblastine, platidiam and bleomycetin. The latter was used in a dose of 10 mg daily intramuscularly or as long-term intravenous infusions. As a result of the treatment complete and partial regression of the tumors was observed in 5 (31 per cent) and 4 (25 per cent) patients, respectively. Leukopenia was the main side effect. By present the total doses of bleomycetin had amounted to 250 mg for 4 patients and to 300-350 mg for 7 patients. No signs of pulmonary toxicity were observed with the use of these doses. The problem of clinical studies of bleomycin analogs is discussed.     

Results of the clinical study of the antineoplastic antibiotic bleomycetin

Bleomycetin was applied to the treatment of 68 patients with common forms of malignant tumors. The objective therapeutic effect was observed in 21 patients (31 per cent). The frequency of the favourable therapeutic effects was the most significant in the group of patients with generalized forms of lymphogranulomatosis: objective remissions for 1 to 4 months and stabilization of the tumor process were attained in 12 (41 per cent) and 8 out of 29 patients, respectively, in 9 patients (31 per cent) treated with bleomycetin progression of the underlying disease was recorded. A less pronounced therapeutic effect (33 per cent of the remissions) was recorded in the patients with nonlymphogranulomatous lymphomas. The use of bleomycetin in 48 out of 68 patients was complicated by certain adverse reactions. Intravenous infusions of bleomycetin in a dose of 10-15 mg twice a week (the total dose up to 125 mg) may be recommended as the initial therapeutic regimen in the oncological practice. The trials have showed that bleomycetin made in the USSR has a sufficiently pronounced activity against lymphogranulomatosis and nonlymphogranulomatous lymphomas. In this respect it is not inferior to the bleomycin analog made in Japan.     

Genetic engineering of Bacillus subtilis for the commercial production of riboflavin

Recombinant DNA engineering was combined with mutant selection and fermentation improvement to develop a strain of Bacillus subtilis that produces commercially attractive levels of riboflavin. The B. subtilis riboflavin production strain contains multiple copies of a modified B. subtilis riboflavin biosynthetic operon (rib operon) integrated at two different sites in the B. subtilis chromosome. The modified rib operons are expressed constitutively from strong phage promoters located at the 5′ end and in an internal region of the operon. The engineered strain also contains purine analog-resistant mutations designed to deregulate the purine pathway (GTP is the precursor for riboflavin), and a riboflavin analog-resistant mutation in ribC that deregulates the riboflavin biosynthetic pathway. Received 22 June 1998/ Accepted in revised form 6 November 1998     

The results of the use of bleomycin and its analogs in the VAB-6 protocol in treating testicular tumors

Efficacy and toxicity of VAB-6 combinations with bleomycin, bleomycetin or peplomycin were studied in treatment of 77 patients with metastases of germ-cell tumors: testicle tumors in 71 patients and extragonadal tumors in 6 patients. After the chemotherapy complete regression was observed in 37 patients (48.7 per cent). In 44 patients (57.1 per cent) residual metastases after the chemotherapy were resected. The frequency of complete regression after using the VAB-6 combinations with bleomycin, bleomycetin and peplomycin amounted to 58.8, 61.5 and 47.1 per cent respectively. The treatment results depended on the disease extent. When the disease extent was minimal complete regression was observed in 87.5 per cent of the patients. The respective figures for the disease moderate and significant extents were 66.7 and 37.8 per cent. During the average observation period of 22.1 months (7-40 months) 39 patients survived and had no signs of the disease. The combinations markedly differed in their toxicity.     

Belomycetin in the combined chemotherapy of testicular tumors

The results of the treatment of 21 patients with testicle tumors of various histological structure, stages III and IV, are presented. The combination of bleomycetin (bleomycin A5), an antitumor antibiotic made in the USSR, with vinblastine and platinum derivatives was used. The antitumor effect was observed in 63 per cent of patients, including 21 per cent of patients with complete regression of tumors. The periods of complete and partial remission were 3--13 and 1--9 months, respectively. The combination is sufficiently effective and may be recommended for the treatment of patients with disseminated malignant tumors of the testicle.     

Antineoplastic antibiotics in the combined chemotherapy of squamous cell carcinoma

Sixty five patients with squamous cell carcinoma of various localization at stages III-IV or with severe relapses were subjected to chemotherapy according to 3 schemes: AMB (adriamycin + methotrexate + bleomycin or bleomycetin), 34 patients; AMBP (adriamycin + methotrexate + bleomycetin + platidiam), 17 patients and AMFP (adriamycin + methotrexate + fluorofur + platidiam), 14 patients. The efficacy of the schemes was 35, 17.7 and 43 per cent respectively. The AMB scheme in treatment of the patients with maxillofacial carcinoma resulted in remission in 8 out of 20 cases (40 per cent). Analysis of the adverse reactions to the chemotherapy showed that all the three schemes were relatively low toxic. The AMB and AMFP schemes may be recommended for treatment of patients with disseminated or inoperable forms of epidermoid tumors in oncological departments.     

In vitro detection of endonuclease IV-specific DNA damage formed by bleomycin in vivo.

Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3'-OH groups that support in vitro synthesis by E.coli DNA polymerase I, to determine whether endonuclease IV-specific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo-) and endonuclease IV-overproducing (p-nfo; approximately 10-fold) strains all supported modest polymerase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis with samples from the nfo- strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and still less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from bleomycin-treated nfo- cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo- strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was > or = 5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxy-pentos-4-ulose residues, but other possibilities are discussed.     

Clinical evaluation of a bleomycin-group antitumor antibiotic bleomycetin

The efficacy of bleomycetin or bleomycin A5 was studied in 128 patients with different malignant neoplasms. The antibiotic was used as a systemic or intracavitary chemotherapeutic agent. Bleomycetin was effective in 75-80, 81.8, 58.3, 70 and 50 per cent of the cases with disseminated derminogenic tumor of the testicle, squamous cell carcinoma of the head and neck, cancer of the penis, carcinoma of the skin and lymphogranulomatosis, respectively. When used intracavitarily the drug was effective in 41.2 per cent of the patients with cancer of the ovaries and lungs, teratoblastoma of the ovaries, cancer of the mammary gland and sarcoma of the soft tissues. Hyperthermia and focal hyperkeratosis as the adverse reactions were observed in 40.6 and 5.4 per cent of the patients, respectively. No toxicity with respect to the lungs was registered.     

Bleomycin, an Antitumor Antibiotic: Improved Microbiological Assay and Tissue Distribution Studies in Normal Mice

A microbiological assay was developed for bleomycin, an antitumor antibiotic reported to be active in human trials. The assay bacterium was a strain of Escherichia coli which is resistant to ethionine. Studies revealed relatively high concentrations of bleomycin in the blood and urine of mice after a single dose, < 0.33 ld(10), injected intraperitoneally.     

Elaboration of the combination of antitumor preparations bleomycetin + 5-fluorouracil + cisplatin

Clinical trials on efficacy and toxicity of combined use of bleomycetin, 5-fluorouracil and cisplatin in patients with disseminated tumor processes were conducted. Two regimens were applied. Regimen I included intravenous administration of cisplatin in a dose of 100-150 mg/m2 on day 1, intramuscular administration of bleomycetin in a dose of 10 mg on days 2-4 and intravenous jet injection of 5-fluorouracil in a dose of 400 mg/m2 on days 2-4. Regimen II consisted of intramuscular administration of bleomycetin in a dose of 10 mg on days 1-3, intravenous jet injection of 5-fluorouracil in a dose of 400 mg/m2 on the same days and intravenous administration of cisplatin in a dose of 100-150 mg/m2 on day 4. The intervals between the courses amounted to 4 weeks. Complete regression of cervical carcinoma relapsing was observed in 1 patient. In 5 patients i.e. 1 with small-cell lung cancer, 3 with squamous cell lung cancer and 1 with metastases of low-differentiated cancer from an undetected focus to supraclavicular lymph nodes the effect was partial. Long-term stabilization of the disease at the background of the treatment for 6-7 months was stated in 3 patients. On the whole the objective response was in 6 out of 22 patients or in 27 per cent. 7 of them were treated with cisplatin in a dose of 150 mg/m2. The regimens of the combined use of 5-fluorouracil, bleomycetin and cisplatin were low toxic. The therapeutic effect showed that the combination was of practical value.     

Visualization of focal nuclear sites of DNA repair synthesis induced by bleomycin in human cells

In this study, we examined DNA repair synthesis in human cells treated with the radiomimetic drug bleomycin, which efficiently induces double-strand breaks (DSBs). Using tyramide-biotin to amplify fluorescent signals, discrete nuclear foci from the incorporation of 5-iododeoxyuridine (IdU) were detected in proliferating human cells treated with bleomycin. We believe this comes from the repair of DSBs. An increase in the number of foci (>5 per nucleus) was detected in a major fraction (75%) of non-S-phase cells labeled for 30 min with IdU 1 h after the end of bleomycin treatment. The fraction of cells with multiple IdU-containing foci was found to decrease 18 h after treatment. The average number of foci per nucleus detected 1 h after bleomycin treatment was found to decrease twofold between 1 and 3.5 h, indicating that the foci may be associated with the slow component of DSB repair. The presence of DSBs in bleomycin-treated cells was confirmed using antibodies against phosphorylated histone H2AX (gamma-H2AX), which is strictly associated with this type of DNA damage. After treatment with bleomycin, non-S-phase cells also displayed heterogeneous nuclear foci containing tightly bound proliferating cell nuclear antigen (PCNA), suggesting an ongoing process of unscheduled DNA synthesis. PCNA is known to be involved in base excision repair, but a fraction of the PCNA foci may also be associated with DNA synthesis occurring during the repair of DSBs.     

Use of catabolic repression in the selection of the glucoamylase-producer Aspergillus niger

The effect of various carbon sources and cAMP on the glucoamylase synthesis in Aspergillus niger was studied to find carbon sources repressed the enzyme synthesis and conditions for the selection of catabolite stable mutants. Maltose at a concentration of 0.5% stimulated the glucoamylase synthesis, but at a concentration of 4% it repressed not only the enzyme synthesis but the growth of the parental strain on the agar medium. The more active mutant 66 was obtained as a result of treatment of Asp. niger st 6 with NG. This mutant is able to grow on the Czapek's medium containing maltose at concentrations 4 or 6%. The mutant 66 produced about 2.9 times more glucoamylase than its parent when maltose was added at 0.5% concentration to the medium. The glucoamylase synthesis in the parental strain was completely repressed under repressing conditions, while the level of the mutant strain activity was 35% from the level of enzyme activity on the medium without the repressor. The addition of cAMP (5.10(-5] resulted in a partial release of maltose (4%) repression of the glucoamylase synthesis in both strains. The results obtained indicate a possibility to select Asp niger mutants with the partially derepressed glucoamylase synthesis. Other regulation mechanisms in addition to catabolite repression may be involved in the regulation of the glucoamylase synthesis.     

Effect of Soviet bleomycin-bleomycetin on the body of animals in multiple parenteral administration]

Toxicity of bleomycetin was studied on 3 animal species (rats, rabbits and dogs). The antibiotic was administered intramuscularly and intravenously in various doses for a prolonged period of time. The death of the rats, rabbits and dogs treated with repeated lethal doses of bleomycetin was due to its toxic effect on the kidneys and probably lungs. The level of urea in the blood of the animals before death increased up to 300--400 mg %. Histological examination of the kidneys revealed the picture of glomerulonephritis. The lungs were highly plethoric and showed areas of alveolar collapse and consolidation consisting mainly of the collapsed alveolar epithelium. The liver was not affected by bleomycetin according to both the results of some functional tests and histological examination. tthe blood sugar level after bleomycetin administration was not altered significantly. The changes in the peripheral blood were not pronounced. An increased P wave, decreased R wave and deep S wave were seen on the ECG. Such deviitions may be due not only to the changes in the myocardium but also to the lung affection. When bleomycetiin was used repeatedly in nonlethal doses (1 mg/kg for rats, 1--2 mg/kg for rabbits and 0.25--0.5 mg/kg for dogs), the above changes were less pronounced or not manifested at all. No inhibitory effect on hemopoiesis is an important positive characteristics of bleomycetin, so that it compares very favourably with most other antitumor drugs.     

In vivo manipulation of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 revealing new insights into its biosynthetic pathway

Bleomycin (BLM), an important clinically used antitumor compound, and its analogs are challenging to prepare by chemical synthesis. Genetic engineering of the biosynthetic pathway in the producer strain would provide an efficient and convenient method of generating new derivatives of this complex molecule in vivo. However, the BLM producing Streptomyces verticillus ATCC15003 has been refractory to all means of introducing plasmid DNA into its cells for nearly two decades. Several years after cloning and identification of the bleomycin biosynthetic gene cluster, this study demonstrates, for the first time, genetic accessibility of this pharmaceutically relevant producer strain by intergeneric Escherichia coli-Streptomyces conjugation. Gene replacement and in-frame deletion mutants were created by lambdaRED-mediated PCR targeting mutagenesis, and the secondary metabolite profile of the resultant mutants confirmed the identity of the BLM biosynthetic gene cluster and established its boundaries. Ultimately, the in-frame blmD deletion mutant strain S. verticillus SB5 resulted in the production of a bleomycin intermediate. The structure of this compound, decarbamoyl-BLM, was elucidated, and its DNA cleavage activity was compared with the parent compounds.     

Biochemical effects of bleomycin A2 on Novikoff hepatoma ascites cells.

Purified nucleolar DNA was markedly degraded at a concentration of 13 mug/ml by bleomycin A2; bleomycin concentrations 20-30 times greater were required to degrade nucleoplasmic DNA. Whole nuclear DNA was degraded to only a small extent at 13 mug/ml but was markedly degraded at higher bleomycin concentrations. Treatment of the various types of DNA with high concentrations of bleomycin A2 produced low molecular weight (approximately 6S) fragments that were no longer sensitive to degradation by bleomycin A2. Hybridization studies demonstrated a loss of ribosomal DNA sequences from nucleolar DNA treated with bleomycin A2 in vitro. Studies on RNA synthesis in Novikoff hepatoma ascites cells in vitro showed there was a decreased uptake of 32Pi into high molecular weight nuclear RNA in the presence of bleomycin A2. These results indicate that nucleolar function is inhibited by a direct effect of bleomycin A2 on nucleolar DNA.     

DNA replication and repair in ataxia telangiectasia cells exposed to bleomycin

A marked increase in sensitivity to bleomycin was observed in two ataxia telangiectasia (AT) lymphoblastoid cell lines compared to that in cell lines from two normal individuals. This sensitivity was obtained at two different concentrations of bleomycin. While normal cells showed a rapid recovery of ability to divide, there was no indication of such a recovery in AT cells up to 120 h after bleomycin treatment. A similar level of breakage of DNA occurred in both cell types after incubation with bleomycin. The rate of repair of these breaks was also the same. DNA synthesis was found to be more resistant to bleomycin in AT cells than in control cells. The latter data are in keeping with results previously obtained using ionizing radiation.     

Regulatory isozymes of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase in the cyanobacterium Anabaena sp. strain ATCC 29151.

Two regulatory isozymes of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase from the cyanobacterium Anabaena sp. strain ATCC 29151 were separated via gel filtration. One isozyme (Mr = 205,000) was sensitive to phenylalanine inhibition, and the other (Mr = 60,000) was sensitive to tyrosine inhibition. The tyrosine-sensitive isozyme from wild-type cells was unstable to gel filtration, whereas the corresponding isozyme from an analog-resistant mutant was insensitive to tyrosine inhibition and was stable.     

The genome-wide DNA sequence specificity of the anti-tumour drug bleomycin in human cells

The cancer chemotherapeutic agent, bleomycin, cleaves DNA at specific sites. For the first time, the genome-wide DNA sequence specificity of bleomycin breakage was determined in human cells. Utilising Illumina next-generation DNA sequencing techniques, over 200 million bleomycin cleavage sites were examined to elucidate the bleomycin genome-wide DNA selectivity. The genome-wide bleomycin cleavage data were analysed by four different methods to determine the cellular DNA sequence specificity of bleomycin strand breakage. For the most highly cleaved DNA sequences, the preferred site of bleomycin breakage was at 5′-GT* dinucleotide sequences (where the asterisk indicates the bleomycin cleavage site), with lesser cleavage at 5′-GC* dinucleotides. This investigation also determined longer bleomycin cleavage sequences, with preferred cleavage at 5′-GT*A and 5′- TGT* trinucleotide sequences, and 5′-TGT*A tetranucleotides. For cellular DNA, the hexanucleotide DNA sequence 5′-RTGT*AY (where R is a purine and Y is a pyrimidine) was the most highly cleaved DNA sequence. It was striking that alternating purine–pyrimidine sequences were highly cleaved by bleomycin. The highest intensity cleavage sites in cellular and purified DNA were very similar although there were some minor differences. Statistical nucleotide frequency analysis indicated a G nucleotide was present at the ?3 position (relative to the cleavage site) in cellular DNA but was absent in purified DNA.