Retinoic acid가 dimethylbenzanthracene과 12-O-tetradecanoyl-phorbol-14-acetate도포에 의한 생쥐 표피세포증식에 미치는 영향

Abstract

[한글]

악성종양은 기본적으로 변형된 세포의 증식으로 인한 것이며 이 변화가 점점 심해지고 진행된 최종 결과가 암 형성이라 할 수 있겠다. 따라서 세포증식을 일으키는 종양촉진의 기전은 피부발암현상에 있어 중요하게 생각되며 촉진제 도포 후 일정 기간 내에 일어나는

일련의 조직변화를 알아봄은 의미있는 것으로 생각된다. 그러나 표피발암의 연구에 이용되는 대표적 종양촉진제 중의하나인 12-O-tetradecanoyl-phorbol-13-acetate(TPA) 관한 연구는 대부분이 장기간 도포한 후의 변화에 관한 것이며 이를 1회 도포한 후에 일어나는

초기의 표피증식의 양상에 대한 연구는 소수이며 잘 알려져 있지 않다. 최근 retinoic acid(RA)가 종양촉진제에 의한 종양형성을 억제한다는 보고들이 있다. RA의 약리학적 작용은 다양하며 이의 작용부위는 다수인 것으로 여겨진다. 또한 피부질환의 RA 치료 중 랑게르한스 세포의 자극이 일어난 보고도 있어 피부 면역방어기전에 중요한 랑게르한스 세포가 RA의 작용부위중의 하나일 가능성이 있다. 발암제에 의해 실험적으로 유발된 병소에는 랑게르한스 세포 수가 감소되어 있으므로, 랑게르한스 세포의 항종양면역반응 및 RA의

작용과 종양촉진제에 의한 세포증식양상과의 관계를 알아봄은 흥미있는 것으로 생각되어 저자는 첫째, 발암제인 7,12-dimethylbenzanthracene(DMBA)을 생쥐 피부에 전 처치한 후

TPA 1회 도포에 의해 초래되는 표피세포의 증식 및 형태학적 변화의 양상을 알아보고, 둘째, 종양형성 억제제로 알려진 RA를 TPA도포 전후에 도포하여 봄으로써 TPA도포에 의한 표피세포의 중식 및 형태학적 변화가 RA에 의해 어떤 영향을 받는지 알아보며, 셋째, 표

피 랑게르한스 세포 수와 RA 및 종양촉진제에 의한 세포중식양상과의 관계를 알아보고자 본 연구를 시도하였다.

실험동물은 근친교배계통 BALB/c 수컷생쥐를 사용하였으며 대조군으로 아세톤 도포군 , DMBA 전처치 후 아세톤 도포군 및 DMBA 전처치 후 RA 도포군, 실험군으로 DMBA 전처치 후 TPA 도포군 및 DMBA 전처치 후 RA 및 TPA 도포군으로 나누어 실험하였다.털을 제거한 생쥐 배부의 피부에 DMBA 400 nmol을 아세톤 0.4 ml에 용해하여 붓으로 도포하였으며 1주일 후에 TPA 40 nmol을 동일 부위에 도포하였고 RA는 TPA도포 1시간 전 및 6시간 후에 34 nmol을 동일 부위에 도포하였으며 대조군은 동량의 아세톤이나 RA를 해당 시기에 도포하였다. 동물의 희생은 TPA도포 후 6시간, 12시간, 24시간, 3일, 1주, 2주에 시행하였으며 bromodeoxyuridine(BrdU)염색을 위해 희생 1시간 전에 BrdU(1 mg/체중 g)를 정맥 주입하였다. 약물도포부위 피부를 절제하여 헤마톡실린-에오진 염색을 시행한 후 광학현미경하에서 표피세포의 변화를 관찰하였으며 표피 mm 당 표피내 핵의 수와 세포분열지수를 산출하였다. 또한 면역과 산화효소법을 이용하여 BrdU염색을 한 후 이의 표지세포지수를 산정하였으며, Ia특이 단세포군 항체를 이용하여 랑게르한스 세포를 염색하고 표피 mm**2당 세포 수를 산출하여 다음과 같은 결과를 얻었다.

1. TPA에 의한 표피의 증식은 TPA도포 후 6시간에 발생되며 대조군에 비해 현저히 증가되었다. 표피내 핵의 수, 세포분열지수 및 표피세포의 BrdU표지지수도 TPA도포 후 6시간에 이미 증가되어 있었으며 이러한 현상은 시간이 지남에 따라 증가하다가 1∼3일을 정점으로 점차 감소하여 2주 후에는 대조군과 비슷한 수준으로 되는 경향을 보였으나 세포분열지수와 BrdU표지지수는 다소 높은 치를 유지하고 있었다.

2. TPA도포에 의한 세포증식은 초기에는 RA 처치에 의해 다소 억제되었으며 후기에 해당되는 1∼2주에는 TPA 도포군이 RA 및 TPA 도포군에-비해 의의있게 높은 상태에 있었다.

3. 각질세포의 손상은 DMBA도포 후 TPA 도포군에 비해 R닐 및 TPA 도포군에서 늦게 시작되었으며 그 정도도 현저히 경하였다. 이러한 현상은 RA의 항염증작용과 관련이 있는 것으로 생각되었다.

4. 랑게르한스 세포 수는 DMBA를 도포한 군에서는 모두 감소되었으나 이는 DMBA도포후 1주경 부터 증가하여 2주에는 아세톤 도포군과 근사한 정도로 회복되었으며 DMBA도포 후 TPA 도포군에 비해 DMBA도포 후 RA및 TPA도포군에서 일찍 회복되었다.

5. TPA를 도포한 군과 도포하지 않은 군간에 세포 증식도에 따른 랑게르한스 세포수의 의의있는 차이는 없었다.

이상의 결과로 보아 TPA 1회 도포에 의한 표피세포의 증식은 RA에 의해 억제되며 발암제 도포 후 감소된 랑게르한스 세포의 회복은 RA에 의해 촉진될 가능성이 있으며 랑게르한스 세포와 표피세포증식과는 무관한 것으로 생각된다.

The effeet of retinoie acid on mousse epidermal eels proliferation produced

by the application of dimethylbenganthracene and 12-0-tetradecanoy1- Phorbo1-

13-acertate

Hyun Chung

Department of Medical Science, The Graduate School, Yonsei Uinversity

(Directed by Associate Professor Kwang Gil Lee)

The principal component of neoplasm is the proliferation of transformed cells.

Cancer formation may be regarded as the end result of progressive, worse

proliferation of the transformed cells. Chemically induced epidermal carcinogenesis

is usually divided into two stapes, initiation and promotion. Initiation involves

the conversion of some epidermal cells into latent neoplastic cells. Promotion

allows for the expression of this neoplastic change. Therefore, important areas7f

investigation are the mechanism of tumor promotion that results in cell

proliferation and a series of early histologic changes produced by the application

of a tumor promoter. The research of epidermal carcinogenesis using

12-0-tetradecanoyl-phorbol-13-acetate (TPA), the most widely used promoting agent,

has focused on its long term effects. Surprising1y, very little is known about the

nature of epidermal hyperplastic growth Produced by a sing1e application of TPA.

Recently, retinoic acid(RA) and certain of its analogues have been shown to

prevent tumor formation in a number of experimental models. RA shows diverse

Pharmacological action, and its action sites seem to vary. Besides, the report of

Langerhans cell stimulation occurring during treatment by RA of skin disease

suggests a possible role of RA on the Langerhans cell activity. As Langerhans cells

are deo1eted in the experimentally induced lesion by chemical carcinogen, it is

very interesting to study the relationship of ceil proliferation by a promoter to

activity of Langerhans cells and the effect of RA. This study was attempted to

investigate the epidermal changes and nature of cell proliferation induced by a

sing1e application of TPA in the skin initiated with 7,

12-dimethylbenzanthraceneDMBA), the effect of RA on the epidermal proliferation,

and the relationship of the number of Langerhans cells to cell proliferation.

One hundred and fifty male inbred BALB/C mice weighing 20∼25g were divided into

5 groups; the acetone applied group, the group applied with acetone after DMBA

initiation, and the group applied with RA after DMBA initiation as control groups,

and the group applied with TPA after DMBA initiation and the group applied with

both RA and TPA after DMBA initiation as test groups. The drugs were applied with a

brush on the dorsal skin after depilation ; 400 nmol of DMBA in 0.4 ml of acetone,

40 nmol of TPA in 0.4 ml of actone, and 34 nmol of RA in 0.4 ml of acetone were

used. TPA was applied 7 days after DMBA application. RA was applied one hour before

and 6 hours after TPA application. Control groups were applied with the same amount

of acetone or RA at the corresponding time. Animals were sacrificed at determined

intervals. One hour before sacrifice, bromodeoxyuridine(BrdU)(1 mg/g) dissolved in

saline was injected via the tail vein for BrdU stain of S phase cells. A 3 × 1 .5

strip of skin was removed. Part of the etrip was used for hematoxylin-eosin and

BrdU stain. The rest was used as an epidermal sheet for Ia antigen staining. BrdU

and la antigen staining was performed by the immunoperoxidase method. Sequential

histologic changes were observed, while also t? counting the number of epidermal

nuclei/mm epidermis, BrdU labelling cells, and mitotic figures, and the number of

Langerhans cells ㎟ epidermis.

The results obtained are as follows:

1. Epidermal hyperplastic growth occurred within hours and increased markedly.

The number of nuclei/mm epidermis, mitotic index, and Brdu labelling index of

epidermal cells increased 6 hours after TPA application. This increase lasted until

day 1 to day 3 after TPA application, and decreased to the level of the control

group after 2 weeks, however, mitotic index and BrdU labelling index were still

higher than the controls.

2. Cell proliferation induced by TPA was inhibited by RA within hours, but at 1-2

weeks, it was significantly higher in the TPA group when compared to the RA plus

TPA group.

3. Damage to keratinocytes began to occur later in the RA plus TPA group than in

the TPA group, and the degree of damage was milder. This is considered to be

related to thea ntiinflammatory action of RA.

4. The number of Langerhans cells was decreased in all the groups initiated with

DMBA. It increased about 2 weeks after DMBA application, reaching the level of the

acetone group. The RA plus TPA group showed earlier recovery in the number of

Langerhans cells.

5. There was no significant difference in the number of Langerhans cells in

relation to epidermal hyperplastic growth between the TPA and the RA Plus TPA

groups.

In conclusion, the results indicate that the epidermal hyperplastic growth

Prpduced by a single application of TPA is inhibited by retinoic acid and is not

related to the number of Langerhans cells. Also the recovery of depleted Langerhans

cells by a chemical carcinogen may be facilitated by retinoic acid.

[영문]

The principal component of neoplasm is the proliferation of transformed cells.

Cancer formation may be regarded as the end result of progressive, worse proliferation of the transformed cells. Chemically induced epidermal carcinogenesis is usually divided into two stapes, initiation and promotion. Initiation involves

the conversion of some epidermal cells into latent neoplastic cells. Promotion allows for the expression of this neoplastic change. Therefore, important areas7f investigation are the mechanism of tumor promotion that results in cell proliferation and a series of early histologic changes produced by the application of a tumor promoter. The research of epidermal carcinogenesis using 12-0-tetradecanoyl-phorbol-13-acetate (TPA), the most widely used promoting agent, has focused on its long term effects. Surprising1y, very little is known about the

nature of epidermal hyperplastic growth Produced by a sing1e application of TPA.

Recently, retinoic acid(RA) and certain of its analogues have been shown to prevent tumor formation in a number of experimental models. RA shows diverse Pharmacological action, and its action sites seem to vary. Besides, the report of Langerhans cell stimulation occurring during treatment by RA of skin disease

suggests a possible role of RA on the Langerhans cell activity. As Langerhans cells are deo1eted in the experimentally induced lesion by chemical carcinogen, it is very interesting to study the relationship of ceil proliferation by a promoter to

activity of Langerhans cells and the effect of RA. This study was attempted to investigate the epidermal changes and nature of cell proliferation induced by a sing1e application of TPA in the skin initiated with 7, 12-dimethylbenzanthraceneDMBA), the effect of RA on the epidermal proliferation, and the relationship of the number of Langerhans cells to cell proliferation.

One hundred and fifty male inbred BALB/C mice weighing 20∼25g were divided into 5 groups; the acetone applied group, the group applied with acetone after DMBA initiation, and the group applied with RA after DMBA initiation as control groups, and the group applied with TPA after DMBA initiation and the group applied with

both RA and TPA after DMBA initiation as test groups. The drugs were applied with a brush on the dorsal skin after depilation ; 400 nmol of DMBA in 0.4 ml of acetone, 40 nmol of TPA in 0.4 ml of actone, and 34 nmol of RA in 0.4 ml of acetone were used. TPA was applied 7 days after DMBA application. RA was applied one hour before and 6 hours after TPA application. Control groups were applied with the same amount of acetone or RA at the corresponding time. Animals were sacrificed at determined

intervals. One hour before sacrifice, bromodeoxyuridine(BrdU)(1 mg/g) dissolved in saline was injected via the tail vein for BrdU stain of S phase cells. A 3 × 1 .5 strip of skin was removed. Part of the etrip was used for hematoxylin-eosin and BrdU stain. The rest was used as an epidermal sheet for Ia antigen staining. BrdU and la antigen staining was performed by the immunoperoxidase method. Sequential histologic changes were observed, while also t? counting the number of epidermal

nuclei/mm epidermis, BrdU labelling cells, and mitotic figures, and the number of Langerhans cells ㎟ epidermis.

The results obtained are as follows:

1. Epidermal hyperplastic growth occurred within hours and increased markedly.

The number of nuclei/mm epidermis, mitotic index, and Brdu labelling index of epidermal cells increased 6 hours after TPA application. This increase lasted until day 1 to day 3 after TPA application, and decreased to the level of the control

group after 2 weeks, however, mitotic index and BrdU labelling index were still higher than the controls.

2. Cell proliferation induced by TPA was inhibited by RA within hours, but at 1-2 weeks, it was significantly higher in the TPA group when compared to the RA plus TPA group.

3. Damage to keratinocytes began to occur later in the RA plus TPA group than in the TPA group, and the degree of damage was milder. This is considered to be related to thea ntiinflammatory action of RA.

4. The number of Langerhans cells was decreased in all the groups initiated with DMBA. It increased about 2 weeks after DMBA application, reaching the level of the acetone group. The RA plus TPA group showed earlier recovery in the number of Langerhans cells.

5. There was no significant difference in the number of Langerhans cells in relation to epidermal hyperplastic growth between the TPA and the RA Plus TPA groups.

In conclusion, the results indicate that the epidermal hyperplastic growth Prpduced by a single application of TPA is inhibited by retinoic acid and is not related to the number of Langerhans cells. Also the recovery of depleted Langerhans

cells by a chemical carcinogen may be facilitated by retinoic acid.