Spontaneous regression of cutaneous melanoma in sinclair swine is associated with defective telomerase activity and extensive telomere erosion.

Recently we proposed the hypothesis that extensive telomeric association of chromosomes is an early manifestation of cell death and asked whether there are extensive telomeric associations present in metaphases of the spontaneously regressing Sinclair swine cutaneous melanoma (SSCM). Our results indicate that early passage SSCMs, in the accelerated growth phase, do not show telomeric associations but do have numerical and other specific structural abnormalities. However, the same melanoma cell lines at late passages or melanomas obtained from middle- and old-aged Sinclair swine show extensive telomeric associations in the form of dicentric, multicentric, and ring configurations. Such abnormal structures are present mostly in metaphases that are hyperploids. Increasing frequencies of apoptotic bodies were also observed in higher passage tumor cell lines obtained from younger animals or in melanomas obtained from older animals. The polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay shows no detectable telomerase activity in any of these regressing swine melanoma cell lines, neither in normal swine skin fibroblasts nor in nevi. However, the fetal swine (i.e., non-regressing) melanoma cells show telomerase activity. Fluorescence in situ hybridization (FISH) results using the commercially available human telomeric repeat DNA probe indicate a reduction of telomeric signals in metaphase and interphase cells of regressing melanomas. From these observations we conclude that spontaneous regression of SSCM is associated with the loss of telomerase activity and a reduction of telomeric repeats that results in the formation of multicentric and ring configurations. Such abnormal chromosome configurations are lost, following the breakage-fusion-bridge-cycles, and result in extensive DNA fragmentation, as shown by laddering experiments, and, finally, cell death.

Cell-killing by paclitaxel in a metastatic murine melanoma cell line is mediated by extensive telomere erosion with no decrease in telomerase activity.

The purpose of this study was to investigate and compare the effects of paclitaxel and its water-soluble conjugates (sodium-pentetic acid-paclitaxel; polyethylene glycol-paclitaxel, and poly[L-glutamic acid]-paclitaxel) on chromosome morphology and induction of apoptosis in a metastatic murine melanoma cell line (K1735 clone X-21). For this, murine melanoma cells were treated continuously for 72 h with three concentrations (1.2 microM, 2.4 microM, and 4.8 microM) of each of paclitaxel, and conjugates. Another set of cells were pulse-treated at 2.4 microM, 4.8 microM and 9.6 microM concentrations of each of these drugs for 4 h and the recovered cells were examined after 72 h. Control cultures received only the solvents (dimethyl sulfoxide or water). Our results showed a significant increase in the frequencies of telomeric associations, chromosome aberrations, polyploidization, distorted and disintegrated chromosome morphology, and reduced telomeric signal intensity by fluorescence in situ hybridization, in treated cultures as compared to the controls. However, we detected no change in telomerase activity. In addition, the majority of interphase nuclei in treated cells showed apoptotic bodies, with chromatin condensation. These in vitro results suggest that cell death induced by paclitaxel and its water-soluble conjugates is due to the loss of telomeric repeats, as shown by reduced signal flourescence and increased telomeric associations.