Effect of TNF-a and IFN-y on the number of population doublings according to the passage. Each of TNF-a and IFN-y showed inhibitory effect on cultured human keratinocyte (Fig. 1). When added together, they showed more inhibitory effects than any of the single cytokines. These inhibitory effects were more potent in the early passage and became weaker through the following passages. There was no specific effect on HaCaT cell (*: p<0.05, n=3). Because inhibitory effects of TNF-a and IFN-y on the keratinocyte are well known, and considering that cells become senescent according to passage, we can postulate that the effect of these cytokines is more pronounced in the early passage of proliferative young cells.

Fig. 1. When cultured human

Fig. 1. When cultured human keratinocytes were treated with TNF-a and/or IFN-y, it showed inhibitory effect on the prolife­ration rate. Through every pass­age of the TNF-a and/or IFN-y showed synergic effect on pro­liferation inhibition. TNF-a and/ or IFN-y did not show inhibitory effect on HaCaT cells(*: p<0.05, n=3).

The effect of TNF-a and IFN-y on the telomerase activity.
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We evaluated the effect of both cytokines on the telomerase activity through each passage (Fig. 2). Telomerase activity was presented as relative telomerase activity (RTA). To do this, the same number of cells from each passage was collected, and treated with each or both cytokines. The result showed increased telomerase activity on the cells of the early passage (P0, P1). After passage 2, these effects became obscured. This was thought to relate to cellular senescence or differentiation. There was no difference of telomerase activity in the HaCaT cells. TS 8.0 cells were used for negative control according to manufacturer’s direction (*: p<0.05, n=3).

Fig. 2. There was a significant

Fig. 2. There was a significant increase of RTA especially in the early passage (P0, P1). Elevated RTA level was more prominent when cells were treated with TNF-a and TNF-y together. These effects became faint from P2 and RTA level showed no significant differences compared to the control of the same pass­age. In HaCaT cell, the cells treated with TNF-a and/or TNF- Y showed no changes in RTA level(*: p<0.05).

Comparison of effect of TNF-a and/or IFN-y on telomerase activity levels according to passage.

To evaluate the different effect of each cytokine according to the passage, we compared the change of RTA of each passage separately. Fig. 3A shows changes of RTA according to passage of normal control. It showed decreased RTA according to the passage. Fig. 3B-D showed RTA changes by the TNF-a and/or IFN-y. In the presence of TNF-a, RTA increased until P1 and decreased from P2. In the presence of INF-y, it also showed increased RTA in the early passage. These effects seemed to be enhanced when it was stimulated by both TNF-a and IFN-y together (*: p<0.05, n=3). viagra 50 mg

Fig. 3. Comparing

Fig. 3. Comparing the RTA levels of cultured human keratinocyte through the passage, RTA increased until P2. However, after P2, RTA decreased more than the level of the P0. (A) Control. There was a mild increase in telomerase activity on P1. However, it decreased after P2 compared with P0. (B) RTA levels of each passage treated with TNF-a were compared. RTA levels were elevated until P1 than decreased according to passage. (C) RTA levels of each passage treated with IFN-y were compared. There were no significant changes until P3. (D) After combined treatment with TNF-a and INF-a, degree of the changes of RTA were enhanced when compared to the result of the single treatment of TNF-a or INF-a.