Mitomycin C가 인체염색체에 미치는 영향에 관한 연구

Abstract

[한글]

Mitomycine C는 Streptomyces caespitosus에서 추출된 항생 물질로서 미생물에서는 DNA 합성을 저해시키고(Shiba, 1958) 또 분해를 일으키는 것으로 알려져 있다(Kersten과 Rauwen, 1961; Reich등, 1961). 또한 Mitomycin C는 세포분열을 저해시키는 작용이 있다는

보고도 있다(Newell, 1964; Caspersson등, 1965). Merz(1961)는 Vicia 염색체 결실과 재배열(rearrangement)이 이질염색질(heterochromatin)에서만 유의적으로 분포되어 있는 것 같다고 보고하였다. Kobayash(1960)는 Mitomycin C가 쥐(rat)의 종양세포 염색체, 특히 핵인(nucleolus)이 연합된 염색체에서 전위(translocation)를 일으키는 것으로 보여 주었다. 또 Hahn(1973)은 Chinese hamster에서 Mitomycin C에 의한 염색체 이상을 분석하였는데 1번과 2번 염색체의 5와 7부위와 X염색체의 제2차 협착부위에 뚜렷한 영향이 있음을 보고하였다. Nowell(1964), Cohen과 Shaw(1964) 그리고 Shaw와 Cohen(1965)은 Mitomycin C가 배양된 사람의 백혈구의 분열을 억제하고 또 염색체 결실과 교환을 작위적으로(non-random) 유발하며 비상동(non-homologous) 염색체 사이의 상호전위(reciprocal translocation)는 물론 체세포 교차(somatic crossing over)와 같은 과정을 유발시킨다고 보고하였

다. German과 LaRock(1969)는 사람의 이배체섬유아세포(diploid fibroblast)를 Mitomycin C로 처리해서 염색체 결실과 재배열 뿐만 아니라 사방사상(quadriradial) 현상을 일으키는 것을 보여주었다. Morad등(1973)은 사람의 임파구를 재료로 Mitomycin C에 의하여 유

발된 염색체 결실 특히 염색체내교환(interchange)의 분포 상황을 quinacrine mustard banding 방법을 사용하여 보고하였다. 그러나 이들은 Q-banding 방법을 적용하였으면서도 실제로는 C-band를 관찰 기술하였다.

Cohen과 Shaw(1964)와 Nowell(1964)은 보통의 염색방법만을 이용하여 같은 농도로 같은 시간 처리해 주었는데도 서로 상이한 결과를 나타냈으므로 본 연구를 통하여 그것을 다시 한번 정확히 확인하고, 염색체 이상의 다양성을 용이하게 관찰할 수 있는 G-banding (Derets와 Shaw, 1971; Schnedl, 1971) 방법을 적용하여 Morad등(1973)의 연구 결과를 확인함과 동시에 G-banding에서 나타나는 더 상세한 관찰을 시도하고자 하며 그러므로써 Mi

tomycin C로 처리한 사람의 백혈구 염색체에 나타나는 미세한 부분의 이상까지도 분석하고자 본 연구는 시도되었다. Mitomycin C(Sigma)를 TC 199 배양액에서 용해 Moorhead등(1960) 방법으로 희석하여 배양된 정상 백혈구를 몇 실험군으로 나눠서 처리하였다. 제1실험군에서는 배양개시후 24시간째에 0.5㎍/㎖, 1.0㎍/㎖의 최종농도는 각각 1시간 처리한 후 199 배양액으로 처리시약을 제거하고 배양액으로 계속 배양하였다. 제2실험군은 배양 개시후 48시간째에서 0.1, 0.25, 0.5, 1.0㎍/㎖의 최종 농도는 24시간 동안 배양 종료시까지 계속 처리해 주었다. 제3 실험군에서는 배양개시와 함께 0.05, 0.1㎍/㎖의 최종 농

도는 72시간 동안 배양종료시까지 계속 처리하였다. 72시간의 배양이 끝난후 Colcemid(0.5㎍/㎖)로 2시간 처리 공기건조법으로 slide를 제작하였다. G-banding 포본제작은 Seabright(1972)의 trypsin 소화방법의 변법과 Shiraishi와 Yosida(1972)의 urea 소화방법의 변

법을 종합시킨 방법 혹은 urea 소화 방법만 사용하여 5% Giemsa로 염색하였다.

이상과 같은 실험에 의하여 다음과 같은 결과를 얻었다.

1. Mitomycin C에 의해서 억제된 분열율(mitotic rate)을 보면 0.1㎍/㎖의 낮은 농도로 24시간 처리해준 세포에서는 분열율이 대조군(control)에 비해서 억제가 안 되었지만 염색체 이상도 아주 적었다. 1.0㎍/㎖로 1시간이나 24시간 처리한 세포에서는 비교적 강하

게 분열율도 억제되고 염색체 이상도 비교적 많이 유발되었지만 0.5㎍/㎖ 농도는 24시간 처리한 세포는 분열율도 저해되고 가장 많은 분석할 만한 이상도 보여 주었다.

2. 이상형의 반 이상을 차지하고 있는 염색체내교환(interchange)은 대부분이 1번, 9번과 16번 염색체였고 적어도 이 염색체들 중 하나의 제2차 협착 부위와 관련되어 있었다.

이 밖에 염색분체(chromatid)와 염색체(chromosome) 결실, 전위형(translocation), 부수체접속(satellite association)등의 이상을 발견되었다.

이상의 실험성적으로 보아 Mitomycin C는 1번, 9번과 16번 염색체의 제2차 협착 부위에 선택적으로 결손을 일으키는 효과적 화학물질이라고 할 수 있다.

Mitomycin C의 농도와 처리시간의 길이 및 배양기간동안의 노출시기는 염색체 이상을 유발하는 아주 중요한 요인들이며 이 요인들은 상이한 유전적 구조를 지니고 있는 개인의 백혈구에 따라 변이를 보이고 있다고 본다.

The effect of mitomycin C on the human chromosomes with G-banding patterns

Tae Sang Tchun

Department of Medical Science, The Graduate School, Yonsei University

(Direct by Professor: Dong Sik Kim, Mo.D.)

Introduction

Mitomycin C is a chemically reactive antibiotics derived from Streptomyces

caespitosus. The drug, is also one of the important carcinostatic antibiotics in

clinical use.

The primary action of mitomycin C has been considered to inhibit the DNA

synthesis of growing cells but does not effect the synthesis of RNA or

protein(Shiba et al., 1959; Greenberg et al., 1961; Boyce and Howard-Flanders,

1964). the effect of the chemical on other bacteria(Levin, 1961; Okubo and Romig,

1966; Iyer, 1966), plant cells(Merz, 1961), cultured animal cells(Kobayashi, 1960;

Shatkin et al., 1961; Hartley-Asp and Kihlman, 1971; Hahn, 1973) and human

chromosomes(Cohen and Shaw, 1964; Nowell, 1964; Shaw and Cohen, 1965; Doi et al.,

1971; Morad et al., 1973) have been noted by many investigators.

The mechanism of its inhibiting action was studied by Iyer and Szybalski(1964)

and Szybalski and Iyer(1964). They proposed the bifunctional alkylating action of

mitomycin C which induced a crosslinking the complementary strands of DNA, which

would inhibit the DNA replication through disturbing the seperation of

double-strands of DNA. Also Kodama(1967) reported the studies of the interaction of

mitomycin C with deoxyribonucleic acid in vitro to help clear the mechanism of

action of mitomycin C.

Cohen and Shaw(1964) and Nowell(1964) described that mitomycin C inhibits mitosis

and causes break and exchanges in chromosomes of cultured human leukocytes. They

reported that the distribution of breaks were nonrandom with a marked excess of

breaks in the secondary constriction regions of chromosomes number 1, 9 and 16.

Again Shaw and Cohen(1965) showed that a process analogous to somatic crossing over

as well as reciprocal translocation between non-homologous autosomes have induced

by mitomycine C. Specially they noted the breaks and rearrangements in the proximal

heterochromatin of the chromosomes and in the secondary constriction of autosomes

number 1 and 9. In 1973, Morad et al., reported the distribution of mitomycin C

induced breaks on human lymphocytes by quinacrine mustard chromosome banding

technique. Although this technique is for Q-bands, they described as C-bands. They

indicated that the secondary constriction of chromosome 1, 9 and 16 involves

intercnhage aberrations.

Since studies by Cohen and Shaw(1964) and Nowell(1964) conducted by conventional

chromosome preparation only, and they also showed different results with same

concentration and same length of treatment of mitomycin C, the present study was

undertaken in order to clear the right dosage and exposure times and also attempt

to confirm the Morad et al.'s results(1973) by G-banding technique which give

identification of various chromosome aberrations easily(Drets and Shaw, 1971;

Schnedl, 1971) and thus, it can be perform detailed analysis of the location of

induced chromosome breakage point s in human leukocyte chromosome after treated

with mitomycin C. Thus, it is hope that this study will provide the basis for

evaluating known or unknown drugs with regard to their specific effects on specific

sites and regions by the identification of specific breakage points in specific

chromosomes if the human complement.

Materials and Methods

Mitomycin C was obtained fro Sigma chemical Co., St. Louis, Mo. U.S.A. The

chemical was diluted in 199 cuture medium. Various concentrations were added to

normal human leukocyte cultures which had been prepared according to a method

similar to that of Moorhead et al.(1960). Control and experimental culture were

prepared in TC chromosome medium(DIFCO) and incubated for 72 hours at 37℃, and

during the incubation mitomycin C was added to a final concentration of 0.05, 0.1,

0.25, 0.5, 1.0ug/ml to culture fluid for different exposure periods. The culture

were harvested after 2 hours of colcemid(final concentration on 0.5ug/ml). Slides

were prepared using 3 part methanol/1 part acetic acid fixation after hypotonic

solution treated, with the air-drying technique of Saksela and Moorhead(1962), and

stained with Giemsa blood stain(Haleco). Urea treatment G-banding was obtained

according to a modification of the technique reported by Shiraishi and

Yosida(1972). Photomicrographs were made using Kodak high contrast copy film in

Nikon photomicroscope.

Results and Conclusion

The mitomycin C was introduced to human leukocyte cultures to investigate the

analysis of the chromosome aberration by G-banding patterns. The relationship

between mitomycine C and secondary constriction; Length of treatment of exposure

time of the compound; and G-banding pattern were discussed. The results are

summarized as follows:

1. The mitotic rate of cultures exposed to mitomycine C at a concentration of 0.1

ug/ml for last 24 hours did not inhibit significantly from that of the control, and

chromosome aberration were relatively very low. However, the mitotic index of cells

exposed 1.0 ug/ml for 1 hour or 24 hours were significantly below the control index

and a relatively high number of aberrations were found. With the concentration of

0.5 ug/ml for last 24 hours of cultures treatment proved to be the most efficient

combination for examining the effect of this compound on human leukocyte chromosome

in this study.

2. Mitomycin C did not appear to break the chromosome randomly. More than half of

the aberrations were interchange type, which were most cases involved with

chromosomes 1, 9 and 16. They are joined in, at least, secondary constriction

regions of one of the those chromosomes. The secondary constriction regions contain

60 to 70 per cent of total breaks of each of those chromosomes. The other types of

chromosome aberrations were simple chromatid and chromosome breaks at the

interother types of chromosome aberration were simple chromatid and chromosome

breaks at the interband of G-bands and translocations. The frequent association of

satellite with 2 to 5 acrocentric chromosome was also observed.

In conclusion, it can be stated that mitomycin C is very specific in causing

lesions to appear at the secondary constrictions 1, 9 and 16.

The dosage of mitomycin C and length of treatments and exposure time during the

culture period are very important factors for induction of chromosome

abnormalities. which vary with the individual leukocytes that have different

genetic constitution.

[영문]

Mitomycin C is a chemically reactive antibiotics derived from Streptomyces caespitosus. The drug, is also one of the important carcinostatic antibiotics in clinical use.

The primary action of mitomycin C has been considered to inhibit the DNA synthesis of growing cells but does not effect the synthesis of RNA or protein(Shiba et al., 1959; Greenberg et al., 1961; Boyce and Howard-Flanders, 1964). the effect of the chemical on other bacteria(Levin, 1961; Okubo and Romig,

1966; Iyer, 1966), plant cells(Merz, 1961), cultured animal cells(Kobayashi, 1960; Shatkin et al., 1961; Hartley-Asp and Kihlman, 1971; Hahn, 1973) and human chromosomes(Cohen and Shaw, 1964; Nowell, 1964; Shaw and Cohen, 1965; Doi et al., 1971; Morad et al., 1973) have been noted by many investigators.

The mechanism of its inhibiting action was studied by Iyer and Szybalski(1964) and Szybalski and Iyer(1964). They proposed the bifunctional alkylating action of mitomycin C which induced a crosslinking the complementary strands of DNA, which

would inhibit the DNA replication through disturbing the seperation of double-strands of DNA. Also Kodama(1967) reported the studies of the interaction of mitomycin C with deoxyribonucleic acid in vitro to help clear the mechanism of

action of mitomycin C.

Cohen and Shaw(1964) and Nowell(1964) described that mitomycin C inhibits mitosis and causes break and exchanges in chromosomes of cultured human leukocytes. They reported that the distribution of breaks were nonrandom with a marked excess of breaks in the secondary constriction regions of chromosomes number 1, 9 and 16.

Again Shaw and Cohen(1965) showed that a process analogous to somatic crossing over as well as reciprocal translocation between non-homologous autosomes have induced by mitomycine C. Specially they noted the breaks and rearrangements in the proximal

heterochromatin of the chromosomes and in the secondary constriction of autosomes number 1 and 9. In 1973, Morad et al., reported the distribution of mitomycin C induced breaks on human lymphocytes by quinacrine mustard chromosome banding

technique. Although this technique is for Q-bands, they described as C-bands. They indicated that the secondary constriction of chromosome 1, 9 and 16 involves intercnhage aberrations.

Since studies by Cohen and Shaw(1964) and Nowell(1964) conducted by conventional chromosome preparation only, and they also showed different results with same concentration and same length of treatment of mitomycin C, the present study was

undertaken in order to clear the right dosage and exposure times and also attempt to confirm the Morad et al.'s results(1973) by G-banding technique which give identification of various chromosome aberrations easily(Drets and Shaw, 1971;

Schnedl, 1971) and thus, it can be perform detailed analysis of the location of induced chromosome breakage point s in human leukocyte chromosome after treated with mitomycin C. Thus, it is hope that this study will provide the basis for evaluating known or unknown drugs with regard to their specific effects on specific sites and regions by the identification of specific breakage points in specific chromosomes if the human complement.

Materials and Methods

Mitomycin C was obtained fro Sigma chemical Co., St. Louis, Mo. U.S.A. The chemical was diluted in 199 cuture medium. Various concentrations were added to normal human leukocyte cultures which had been prepared according to a method similar to that of Moorhead et al.(1960). Control and experimental culture were

prepared in TC chromosome medium(DIFCO) and incubated for 72 hours at 37℃, and during the incubation mitomycin C was added to a final concentration of 0.05, 0.1, 0.25, 0.5, 1.0ug/ml to culture fluid for different exposure periods. The culture

were harvested after 2 hours of colcemid(final concentration on 0.5ug/ml). Slides were prepared using 3 part methanol/1 part acetic acid fixation after hypotonic solution treated, with the air-drying technique of Saksela and Moorhead(1962), and

stained with Giemsa blood stain(Haleco). Urea treatment G-banding was obtained according to a modification of the technique reported by Shiraishi and Yosida(1972). Photomicrographs were made using Kodak high contrast copy film in Nikon photomicroscope.

Results and Conclusion

The mitomycin C was introduced to human leukocyte cultures to investigate the analysis of the chromosome aberration by G-banding patterns. The relationship between mitomycine C and secondary constriction; Length of treatment of exposure

time of the compound; and G-banding pattern were discussed. The results are summarized as follows:

1. The mitotic rate of cultures exposed to mitomycine C at a concentration of 0.1 ug/ml for last 24 hours did not inhibit significantly from that of the control, and chromosome aberration were relatively very low. However, the mitotic index of cells

exposed 1.0 ug/ml for 1 hour or 24 hours were significantly below the control index and a relatively high number of aberrations were found. With the concentration of 0.5 ug/ml for last 24 hours of cultures treatment proved to be the most efficient

combination for examining the effect of this compound on human leukocyte chromosome in this study.

2. Mitomycin C did not appear to break the chromosome randomly. More than half of the aberrations were interchange type, which were most cases involved with chromosomes 1, 9 and 16. They are joined in, at least, secondary constriction regions of one of the those chromosomes. The secondary constriction regions contain

60 to 70 per cent of total breaks of each of those chromosomes. The other types of chromosome aberrations were simple chromatid and chromosome breaks at the interother types of chromosome aberration were simple chromatid and chromosome breaks at the interband of G-bands and translocations. The frequent association of satellite with 2 to 5 acrocentric chromosome was also observed.

In conclusion, it can be stated that mitomycin C is very specific in causing lesions to appear at the secondary constrictions 1, 9 and 16.

The dosage of mitomycin C and length of treatments and exposure time during the culture period are very important factors for induction of chromosome abnormalities. which vary with the individual leukocytes that have different genetic constitution.