Differentiation of human malignant melanoma cells that escape apoptosis after treatment with 9-nitrocamptothecin in vitro

Abstract

After in-vitro exposure to 0.05 micromol/L 9-nitrocamptothecin (9NC) for periods of time longer than 5 days, 65% to 80% of the human malignant melanoma SB1B cells die by apoptosis, whereas the remaining cells are arrested at the G2-phase of the cell cycle. Upon discontinuation of exposure to 9NC the G2-arrested cells resume cell cycling or remain arrested depending on the duration of 9NC exposure. In contrast to cycling malignant cells, the cells irreversibly arrested at G2 exhibit features of normal-like cells, the melanocytes, as assessed by the appearance of dendrite-like structures; loss of proliferative activity; synthesis of the characteristic pigment, melanin; and, particularly, loss of tumorigenic ability after xenografting in immunodeficient mice. Further, the expression of the cyclin-dependent kinase inhibitor p16 is upregulated in the 9NC-treated, G2-arrested, but downregulated in density G1-arrested cells, whereas the reverse is observed in the expression of another cyclin-dependent kinase inhibitor, p21. These results suggest that malignant melanoma SB1B cells that escape 9NC-induced death by apoptosis undergo differentiation toward nonmalignant, normal-like cells.

Figure 1

Photomicrographs of SB1B melanoma cells exposed to 9NC. SB1B malignant melanoma cells were exposed to 0.05 µmol/L 9NC for various periods of time before they were stained and photomicrographed. The cells received no treatment (A) or were treated with 9NC for 24 hr (B), 7 days (C), and 14 days (D). (Arrows indicate dendritic extensions of the cells. Bar equals 100 µm in D).

Figure 2

Flow cytometry analysis of SB1B cells treated with 9NC. Untreated (A) and 9NC-treated (B–J) cells were subjected to flow cytometry analysis of the cell cycle. The treatment period was 6 hr (B), 12 hr (C), 24 hr (D), 3 days (E), 7 days (F), 14 days (G), 21 days (H), 30 days (I), and 37 days (J). Only trypsin-detached cells were subjected to analysis. (G₀ = G₀+G₁ cells; G₂ = G₂+M cells; Ap, apoptotic cells. Arrowheads indicate histogram fractions representing hyperdiploid cells).

Figure 3

Cell cycle analysis of 9NC-released cells. Cells were treated with 0.05 µmol/L 9NC for 10 days, then transferred to fresh media in absence of 9NC. After release from 9NC treatment, cells were collected at desired times and subjected to cell cycle analysis by flow cytometry. The 9NC-released cells were collected at 6 hr (A), 12 hr (B), 24 hr (C), 3 days (D), 10 days (E), and 21 days (F). For designations of histogram cell fractions, see Figure 2.

Figure 4

Photography of cell pellets. Trypsin-detached cells were pelleted by gentle centrifugation, stored in formalin, and photographed. The pellets shown were from cultured cells that received no treatment (A), those cells continuously exposed to 9NC for 24 hr (B), 3 days (C), 7 days (D), 14 days (E), 21 days (F), 30 days (G), and 37 days (H); and those cells that were exposed to 9NC for 21 days, then released from 9NC treatment for 24 hr (I), 4 days (J), 14 days (K), and 21 days (L).

Figure 5

Detection of p16 and p21 by Western blot analysis. Whole cell extracts were prepared, and samples containing equal protein concentrations were subjected to Western blot analysis. Presence of p16 and p21 was visualized by chemiluminescence. The extracts were prepared from SB1B cells that received no treatment, or were continuously exposed to 0.05 µmol/L 9NC for 1, 3, 14, and 21 days (lanes under the indication: days of 9NC treatment) or after 12 days of 9NC treatment were transferred to and remained in 9NC-free medium for 1, 4, 14, and 21 days (lanes under the indication: days after 9NC release).