Bleomycin이 인체 염색체에 미치는 영향에 관한 실험적 연구

Abstract

[한글]

Bleomycin은 Streptomyces verticillus에서 추출된 항생-항암물질로서 squamous cell carcinoma, testicular tumor와 malignant lymphoma등의 치료에 사용되어 좋은 효과를 얻고 있으며 특히 조혈조직이나 면역기능을 억제하지 않아 다른 항암제와 병용할 수 있는 큰 장점이 있다.

지금까지의 연구 결과 bleomycin은 RNA와 단백질 합성에는 영향을 미치지 않으며 DNA에만 선택적으로 작용하고 그 결과 염색체의 수나 구조적 이상을 유발하는 것으로 알려져 있다.

그러나 염색체에 미치는 영향에 관한 연구에 있어 실험방법과 관찰범위가 서로 다르고 불안전하여 아직도 작용 양상과 특이성에 확실한 정설이 없으며 더욱이 세포분열에 미치는 영향에 관해서는 거의 보고된 것이 없어 그 작용기전의 추구가 요원한 실정이다.

따라서 본 연구는 배양된 인체 백혈구에 bleomycin의 농도와 처리시간을 광범위하고 타당성 있게 변화 작용시켜 세포분열율의 변화와 염색체의 구조적 변화의 양상과 특이성을 관찰하여 작용기전의 일단과 작용시기를 추구하고자 시행하였으며 염색체 표본을 최근 염

색체 동정과 변화관찰에 정확하고 신뢰성이 있다고 인정되고 있는 G-banding 술식을 병용하여 일부는 단순 Giemsa 염색법으로, 일부는 trypsin-Giemsa 혹은 urea trypsin-Giemsa 염색법으로 제작하였다. 배양백혈구에 bleomycin을 작용시킨 실험군은 bleomycin농도와

처리 시간에 따라 세분하였는데 제 1 실험군은 10㎍/ml 24시간 처리 하였고 제 2 실험군은 25㎍/ml로 24시간 처리하였으며 제 3과 제 4실험군은 50㎍/m1건 각각 24시간과 18시간 처리하였다.

대조군을 포함한 각 실험군에서 세포분열율을 산출하고 중기분열세포는 현미경하 사진촬영 및 핵형도 작성으로, 또는 eye-karyotyping으로 분석을 시행하여 다음과 같은 결과를 얻었다.

1. Bleomycin을 10㎍/m1로 24시간 처리한 경우에 배양백혈구의 세포분열은 가장 효과적으로 억제되었으며 세포분열율의 변화는 염색체의 구조적 이상 유발빈도의 변화보다 더욱 급격하고 현저하였다.

2. Bleomycin으로 인한 염색체의 구조적 이상의 관찰분석에 가장 적합한 처리농도와 시간은 10∼25㎍/ml로 24시간 처리한 경우이었으며 그 구조적 이상율은 18.16∼28.09%로서 대조군의 6.15%에 비해 현저한 증가를 보였다. 그 이상의 농도증가와 처리시간의 다과에서는 다소의 변동만을 보였다.

3. 염색체의 구조적 이상형의 대부분은 염색분체형으로 나타났으며 절단이 가장 많았고 다음으로 결손, 전위, 파편, 이중심절염색체와 환염색체의 순으로 발생하였다.

4. 염색체의 구조적 이상은 1-3번 염색체와 4-5번 염색체에 가장 호발하였고 다음으로 6-12번 염색체와 X염색체에서 나타났다. 각 염색체간에서 그 이상형의 종류와 빈도의 분포에 큰 차이가 없었다.

이상의 결과를 종합하여 보면. 항암제인 bleomycin은 세포분열을 직접적으로 저지할 뿐 아니라 DNA합성기를 전후하여 염색체의 구조에도 영향을 미쳐서 구조적 이상을 유발시켜 세포의 계대증식을 저해하는 것으로 생각된다.

The effect of Bleomycin on Human Chromosomes

Seung Kang Choi

Department of Medical Science, The Graduate School, Yonsei, University

(Directed by Professors: Chong Soon Wang, M.D. and Tai Sun Shin, M.D.)

Bleomycin, a new antitumor antibiotic elaborated by Streptomyces verticillus, has

shown efficacy against squamous cell carcinomas, testicular neoplasms and malignant

lymphoma. In contrast to the effects of other cytotoxic chemotherapeutic agents,

depression of the hematopoietic and immune system has only rarely teen reported and

so the potential exists for the use of b1eomycin in combination with other agents

without increasing the toxicity of such agents.

As reported in previous papers, bleomycin reacts with DNA selectively and

consequently the development of chromosome aberrations. numerical and structural,

has been reported in mammalian cells cultured in a medium with bleomycin.

However, few reports have dealt with the effects of bleomycin on mitosis, and

chromosome aberrational patterns, its specific character and time of action are now

to be studied because of scanty volume and variable results of previous

experimental or clinical studies

Thus, this study was undertaken to evaluate the effects of this new drug on

mitosis and chromosomes of human leukocytes, especially on the chromosome

aberrational patterns and their specific character.

In the present investigation, human leukocyte cultures were divided into 2

groups, control and treated, and then the treated group was divided into 4 groups

according to concentration and duration of bleomycin, as fellows; Group 1,

bleomycin was administered at a concentration of 10㎍/ml for 24 hours: Group 2, at

a concentration of 25㎍/m1 for 24 hours; Group 3, at a

concentration of 50㎍/m1 for 24 hours; and Group 4, at a concentration of 50 ㎍

/ml for 18 hours.

The chromosome slides were stained with either a simple Giemsa or trypsin- or

urea trypsin-Giemsa banding technique. Trypsin or urea trypsin treated G-banding

was obtained according to a modification of the technique reported by Seabright

(1971).

In each group mitotic rates were calculated and chromosome aberrations were

observed with regards to prevalences, patterns and bleomycin specific effects by

paper karyotyping with protomicrographs or eye karyotyping.

The results obtained are summerized as follows:

1. The concentration of 10 ㎍/ml for 24 hours of culture treatment proved to be

the most efficient combination for examining the effects of bleomycin on the

mitosis, which showed an acute and significant decline of mitotic rate.

The bleomycin effects on the mitosis proved to be stronger than on the induction

of chromosome aberration.

2. For examining the effects of bleomycin on human leukocyte chromosomes the

concentration of 10∼25㎍/ml for 24 hours was the most efficient combination, in

which the structural aberrational rates were 18.16∼28.09%.

The prevalence of chromosome aberration gradually increased up to the

concentration of 25 ㎍/ml, but little change was observed when treated with the

higher concentrations.

3. The chromosome aberration in bleomycin treated groups was of the chromatid

type in the majority. Among the patterns of chromosome aberration, break made up

about half the incidence in each group, followed by deletion, translocation,

fragmentation, dicentric and ring chromosome in that order.

4. The chromosome aberration in bleomycin treated groups was mast frequently

observed in the chromosomes, number 1∼3 and 4∼5 and then the chromosomes, number

6∼12 and sex chromosome X. But there was no recognizable correlation between

chromosome numbers and chromosome aberrational patterns.

From this result, it is suggested that the anticancer drug, bleomycin, inhibits

cell progression into mitosis directly and cell propagation by induction of

chromosome aberration in pre-, and post-DNA synthetic resting periods.

[영문]

Bleomycin, a new antitumor antibiotic elaborated by Streptomyces verticillus, has shown efficacy against squamous cell carcinomas, testicular neoplasms and malignant lymphoma. In contrast to the effects of other cytotoxic chemotherapeutic agents, depression of the hematopoietic and immune system has only rarely teen reported and so the potential exists for the use of b1eomycin in combination with other agents without increasing the toxicity of such agents.

As reported in previous papers, bleomycin reacts with DNA selectively and consequently the development of chromosome aberrations. numerical and structural, has been reported in mammalian cells cultured in a medium with bleomycin.

However, few reports have dealt with the effects of bleomycin on mitosis, and chromosome aberrational patterns, its specific character and time of action are now to be studied because of scanty volume and variable results of previous experimental or clinical studies

Thus, this study was undertaken to evaluate the effects of this new drug on mitosis and chromosomes of human leukocytes, especially on the chromosome aberrational patterns and their specific character.

In the present investigation, human leukocyte cultures were divided into 2 groups, control and treated, and then the treated group was divided into 4 groups according to concentration and duration of bleomycin, as fellows; Group 1, bleomycin was administered at a concentration of 10㎍/ml for 24 hours: Group 2, at a concentration of 25㎍/m1 for 24 hours; Group 3, at a

concentration of 50㎍/m1 for 24 hours; and Group 4, at a concentration of 50 ㎍ /ml for 18 hours.

The chromosome slides were stained with either a simple Giemsa or trypsin- or urea trypsin-Giemsa banding technique. Trypsin or urea trypsin treated G-banding was obtained according to a modification of the technique reported by Seabright (1971).

In each group mitotic rates were calculated and chromosome aberrations were observed with regards to prevalences, patterns and bleomycin specific effects by paper karyotyping with protomicrographs or eye karyotyping.

The results obtained are summerized as follows:

1. The concentration of 10 ㎍/ml for 24 hours of culture treatment proved to be the most efficient combination for examining the effects of bleomycin on the mitosis, which showed an acute and significant decline of mitotic rate.

The bleomycin effects on the mitosis proved to be stronger than on the induction of chromosome aberration.

2. For examining the effects of bleomycin on human leukocyte chromosomes the concentration of 10∼25㎍/ml for 24 hours was the most efficient combination, in which the structural aberrational rates were 18.16∼28.09%.

The prevalence of chromosome aberration gradually increased up to the concentration of 25 ㎍/ml, but little change was observed when treated with the higher concentrations.

3. The chromosome aberration in bleomycin treated groups was of the chromatid type in the majority. Among the patterns of chromosome aberration, break made up about half the incidence in each group, followed by deletion, translocation, fragmentation, dicentric and ring chromosome in that order.

4. The chromosome aberration in bleomycin treated groups was mast frequently observed in the chromosomes, number 1∼3 and 4∼5 and then the chromosomes, number 6∼12 and sex chromosome X. But there was no recognizable correlation between chromosome numbers and chromosome aberrational patterns.

From this result, it is suggested that the anticancer drug, bleomycin, inhibits cell progression into mitosis directly and cell propagation by induction of chromosome aberration in pre-, and post-DNA synthetic resting periods.