종양세포유전학의 선구자들

 Peter C. Nowell   Janet D. Rowley  Alfred G. Knudson, Jr

 


Peter C. Nowell 박사가 필라델피아 염색체를 발견한 것은 1960년이었다. 오늘날의 기준으로는 당시 염색체연구는 매우 원시적이었고, 1956년에야 인체 염색체수는 46개라는 것이 밝혀졌다. 염색체를 현미경하에서 사진촬영한 후 하나하나 짤라내고 크기순으로 배열하여 핵형을 밝혀내었다. 펜실바니아의대 병리학교실의 종양생물학자인 Nowell은 당시 아무런 증거는 없었지만 종양과 유전자 변이의 관련성에 관심을 갖고 있었다.

어느 날 Nowell은 동료인 David Hungerford와 같이 배양중인 백혈병세포에서 세포분열 양상과 특수염색으로 많은 염색체를 관찰할 수 있었다. 또한 만성골수성백혈병 종양세포에 조그마한 비정상 염색체가나타난다는 놀라운 사실도 발견하게 되었다.

당시 종양의 유전적 근거에 대해 별다른 생각들이 없었던 때  Nowell 연구결과는 단일 염색체내 특정 유전결함으로 인해 결함세포가 축적되고 종양형성 세포군으로 이어진다는 첫 번째 명백한 증거가 되었다. Nowell과 Hungerford가 발견한 필라델피아 염색체란 그들이 일하던 도시 이름을 딴 것이며, 이 조그만 염색체는 골수계 종양인 CML의 일정한 표지자가 되었다. 그런데 당시 많은 학자들은 염색체 이상이 종양의 결과이지 원인은 아니라고 믿었다. 그 후 다른 종양에서 일정한 양상의 염색체 이상이 발견되기까지는 10년이란 세월이 흘렀고, 또한 필라델피아 염색체가 작아진 이유를 정확하게 이해하는데도 10년 이상이 걸렸다.

  

여기에 그림을 넣으십시오

Peter C. Nowell, M.D.
Professor of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylania

 

Janet D. Rowley, M.D.은 1961년 남편을 따라 영국 옥스포드에서 잠깐 머무런 적이 있지만 대부분의 시간을 시카고대학에서 보내면서 연구경력을 쌓았다. 처음에는 혈액질환에서의 염색체 이상에 대해 별다른 관심이 없었지만 백혈병세포의 염색체 관찰에 몰두하면서 흥미를 가지게되었다.

 CML 환자에서 22번 염색체 일부가 9번 염색체로 이동하을 밝혔다. 게다가 9번 염색체의 일부 영역이 발암유전자를 가지고 22번 염색체 절단점으로 이동하는데, Rowley는 종양에서의 염색체 전좌형이 CML 원인임을 명백한 증거 제시로 밝힌 첫뻔째 학자였다. Rowley는 연달아 AML의 특징을 규정할 수 있는 여러가지 염색체 전좌형을 발견하였다. 최근에는 비정상 염색체를 찾아내는 spectral karyotype라는 검사기법을 사용하여 소아백혈병의 염색체 재배열에 관한 연구를 계속하고 있다.

 

 

Janet D. Rowley, M.D., D.Sc.
Professor of Medicine and of Molecular Genetics and Cell Biology, University of Chicago Medical Center, University of Chicago, Chicago, Illinois

 

Explaining why some tumors are hereditary and others appear to be "sporadic" was one of the great conundrums of cancer biology until Alfred G. Knudson, Jr., M.D., Ph.D., came up with the "two-hit" hypothesis that provided a unifying model for understanding cancer that occurs in individuals who carry a "susceptibility gene," and cancers that develop because of randomly induced mutations in otherwise normal genes. Like many significant conceptual leaps in science, Knudson's "two-hit" hypothesis was met with skepticism when he first published it in 1971.

Knudson, who has been affiliated with the Fox Chase Cancer Center in Philadelphia since 1976, was studying children with retinoblastoma, a cancer of the eye, noting differences between the 40 percent of cases with heritable tumors and the 60 percent of non-heritable cases. "Most people assumed that retinoblastoma genes were inherited in a dominant fashion - that is, if you had the gene, you would get the cancer," Knudson said. But he observed the variable number of tumors that develop in individuals who inherit one retinoblastoma gene, and proposed that a second mutation, after conception of the child, was necessary for a tumor to develop. The same gene, known as RB1, is involved in children with the non-hereditary form, but both mutations, or "hits," occur after conception.

The "hits" can occur in many ways - from an environmental toxin, dietary factors, radiation, or the kind of random mutation that sometimes occurs during the intricate process of normal cell replication. Knudson proposed that retinoblastoma develops either because both copies of a key gene are lost, or because they are inactivated and unable to function.

In essence, Knudson, far ahead of his time (and ahead of his own hard data) hypothesized that some genes' normal role in life is to behave as anti-cancer or "tumor-suppressor genes" that keep cell division under healthy control. At first, the strength of his hypothesis rested on a complex mathematical model, but was supported in 1976 when Knudson and others showed that some patients with hereditary retinoblastoma are missing a segment of chromosome 13 in all of their cells. In 1986, other scientists applied the tools of molecular technology to clone the gene, RB1, so that it's function as a tumor-suppressor could be studied in detail.

One of the most significant achievements of molecular genetics in the past few years has been the identification of a number of tumor-suppressor genes that, when mutated, lose their ability to control cell division. Malignancy is the result. Although Knudson's initial studies were directed at relatively rare tumors, including retinoblastoma and Wilm's tumor (another childhood cancer with heritable components), it is now apparent that his two-hit hypothesis explains the etiology or origin of many common forms of cancer, and is one of many defining concepts behind all of modern cancer biology.

 

 

Alfred G. Knudson, Jr., M.D., Ph.D.
Distinguished Scientist and former President, Fox Chase Cancer Center, Philadelphia, Pennsylvania

 

염색체 분염법의 역사

History of Chromosome Banding Techniques

 

Stain or Banding Technique

Investigator

Year

Q-banding

Caspersson, Zech, Johansson

1970

G-banding (by trypsin)

Seabright

1971

G-banding (by acetic-saline)

Sumner, Evans, Buckland

1971

C-banding

Arrighi, Hsu

1971

R-banding (by heat and Giemsa)

Dutrillaux, Lejeune

1971

G-11 stain

Bobrow, Madan, Pearson

1972

Antibody bands

Dev, et al

1972

R-banding (by fluorescence)

Bobrow, Madan

1973

In vitro bands (by actinomycin D)

Shafer

1973

T-banding

Dutrillaux

1973

Replication banding

Latt

1973

Silver (NOR) stain

Howell, Denton, Diamond

1973

High resolution banding

Yunis

1975

DAPI/distamycin A stain

Schweizer, Ambros, Andrle

1978

Restriction endonuclease banding

Sahasrabuddhe, Pathak, Hsu

1978

 

GTL-Banding

오늘날 가장 보편적으로 이용되는 G-분염법(G-Banding)은 GTL-Banding이라고도 불리며 Giemsa/ Trypsin/Leishman banding의 약자인데 1970년 Giemsa 박사에 의해 고안되었다. 이 염색법은 Trypsin을 사용하여 염색체를 구성하는 histone 단백을 부분적으로 소화시키게 되는데, 즉 trypsin에 의해 염색체가 느슨하게 풀어지고 노출된 DNA를 Leishman 염색액으로 염색하는 원리이다. 결과적으로 나타나는 염색대(band)는 각 염색체에 특이하게 나타나고, 같은 쌍의 염색체를 감별할 수 있게된다.


Q           Q-bands

QF         Q-bands by fluorescence

QFQ       Q-bands by fluorescence using quinacrine

QFH       Q-bands by fluorescence using Hoechst 33258

 

G           G-bands

GT         G-bands by trypsin

GTG       G-bands by trypsin using Giemsa

GAG       G-bands by acetic saline using Giemsa

 

C            C-bands

CB          C-bands by barium hydroxide

CBG       C-bands by barium hydroxide using Giemsa

 

R            R-bands

RF          R-bands by fluorescence

RFA        R-bands by fluorescence using acridine orange

RH          R-bands by heating

RHG       R-bands by heating using Giemsa

RB          R-bands by BrdU

RBG       R-bands by BrdU using Giemsa

RBA        R-bands by BrdU using acridine orange

Source

    Priest, JH. Banding Procedures and Special Stains. In The Cytogenetic Symposia, 1994, B. J. Kaplan and K. S. Dale, Editors. The Association of Cytogenetic Technologists, Burbank, California, 1994.


References
  1. Arrighi FE, Hsu TC. Localization of heterochromatin in human chromosomes. Cytogenetics 10:81-86, 1971.
  2. Bobrow M, Madan K, Pearson, PL. Staining of some specific regions of human chromosomes, particularly the secondary constriction of No. 9. Nature New Biol. 238:122-124, 1972.
  3. Bobrow M, Madan K. The effects of various banding procedures on human chromosomes studied with acridine orange. Cytogenetics and Cell Genetics12:145-156, 1973.
  4. Caspersson T, Zech L, Johansson C. Differential banding of alkylating fluorochromes in human chromosomes. Exp Cell Res. 60:315-319, 1970.
  5. Dev VG; Warburton D, Miller OJ, Miller DA, Erlanger BF, Beiser SM. Consistent pattern of binding of anti-adenosine antibodies to human metaphase chromosomes. Exp Cell Res. 74:288-293, 1972.
  6. Dutrillaux B, Lejeune J. Sur une novelle technique d'analyse du caryotype human. C R Acad Sci Paris 272:2638-2640, 1971.
  7. Dutrillaux B. Nouveau systeme de marquage chromosomique: Les bands T. Chromosoma 41:395-402, 1973.
  8. Howell WM, Denton TE, Diamond JR. Differential staining of the satellite regions of human acrocentric chromosomes. Experientia 31:260-262, 1975.
  9. Latt SA. Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc Nat Acad Sci. US 70:3395-3399, 1973.
  10. Sahastrabuddhe CG, Pathak S, Hsu TC. Responses of mammalian metaphase chromosomes to endonuclease digestion. Chromosoma 69:331-338, 1978.
  11. Schweizer D, Ambros P, Andrle M. Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide, antibiotic distamycin A. Exp Cell Res. 111:327-332, 1978.
  12. Seabright M. A reapid banding technique for human chromosomes. Lancet 2:971-972, 1971.
  13. Shafer DA. Banding human chromosomes in culture with actinomycin D. Lancet 1:828, 1973.
  14. Sumner AT, Evans HJ, Buckland RA. A new technique for distinguishing between human chromosomes. Nature New Biol. 232:31-32, 1971.
  15. Yunis JJ, Sanchez O. The G-banded prophase chromosomes of man. Humangenetik 27:167-172, 1975.

Recommended Reading
Hsu, TC. Human and Mammalian Cytogenetics. An Historical Perspective. Springer-Verlag, New York; 1979.

[2001년 7월 4일]