UV-induced ablation of the epidermal basal layer including p53-mutant clones resets UV carcinogenesis showing squamous cell carcinomas to originate from interfollicular epidermis.

Chronic ultraviolet (UV) exposure induces clones of cells overexpressing mutant p53 in the interfollicular (IF) epidermis and subsequently squamous cell carcinomas (SCCs) with similar p53 mutations. Mutated p53 may give cells growth advantage over neighbouring cells by impaired apoptosis. We tested this by UV overexposure of skin laden with p53-mutant clones and assessed the impact on subsequent tumour development. P53-mutant clones were induced in two groups of hairless SKH1 mice by daily exposures (500 J/m(2) UV from TL12 lamps) for 28 days. On day 29, one group was overexposed (to 10 kJ/m(2) UV), whereas the control group received the regular daily dose. After 1 week of recovery, the daily exposures were resumed in both groups to induce SCCs. UV overexposure forced the entire IF basal layer into caspase-3-driven apoptosis while leaving overlying layers with sunburn cells intact. No apparent regions were spared from apoptosis. Pulse-chase BrdU labelling showed the IF epidermis to be repopulated from the hair follicles (remaining p63 positive). One week after overexposure, the p53-mutant clones had virtually disappeared (0.6, 95% confidence interval 0.5-0.8 per mouse versus 102, 59-179, without overexposure). Tumour development was significantly delayed after UV overexposure (P < 0.0001) by an average of 27 days (standard error of the mean 3); a period matching that of daily exposures preceding the overexposure. Thus, we found that UV-induced ablation of the IF epidermal basal layer eliminates p53-mutant clones and resets UV carcinogenesis. Furthermore, and in contrast with earlier reports, our data show that UV-induced p53-mutant clones and SCCs originate from the IF epidermis.

Rapamycin impairs UV induction of mutant-p53 overexpressing cell clusters without affecting tumor onset.

Because of its antitumor effect, the immunosuppressant rapamycin holds great promise for organ transplant recipients in that it may lower their cancer risk. In a mouse model, we showed previously that rapamycin inhibits the outgrowth of primary skin carcinomas induced by UV radiation. However, the tumors that did grow out showed an altered p53 mutation spectrum. Here, we investigated whether this shift in p53 mutations already occurred in the smallest tumors, which were not affected in onset. We found that rapamycin did not alter the mutational spectrum in small tumors and in preceding microscopic clusters of cells expressing mutant-p53. However, rapamycin did reduce the number of these cell clusters. As this reduction did not affect tumor onset, we subsequently investigated whether rapamycin merely suppressed expression of mutated p53. This was not the case, as we could demonstrate that switching from a diet with rapamycin to one without, or vice versa, did not affect the number of existing mutant-p53 expressing cell clusters. Hence, rapamycin actually reduced the formation of mutant-p53 cell clusters. In wild-type and p53-mutant mice, we could not measure a significant enhancement of UV-induced apoptosis, but we did observe clear enhancement in human skin equivalents. This was associated with a clear suppression of HIF1α accumulation. Thus, we conclude that rapamycin reduces the formation of mutant-p53-expressing cell clusters without affecting tumor onset, suggesting that tumors grow out of a minor subset of cell clusters, the formation of which is not affected by rapamycin.

Early and late effects of the immunosuppressants rapamycin and mycophenolate mofetil on UV carcinogenesis.

Increased skin cancer risk in organ transplant recipients has been experimentally emulated with enhanced UV carcinogenesis from administering conventional immunosuppressants. However, newer generation immunosuppressive drugs, rapamycin (Rapa) and mycophenolate mofetil (MMF), have been shown to impair angiogenesis and outgrowth of tumor implants. To ascertain the overall effect on UV carcinogenesis, Rapa and MMF were admixed into the food pellets of hairless SKH1 mice receiving daily sub-sunburn UV dosages. With immunosuppressive blood levels neither of the drugs affected onset of tumors (<2 mm), but in contrast to MMF, Rapa significantly increased latency of large tumors (>or=4 mm, medians of 190 vs 125 days) and reduced their multiplicity (1.6 vs 4.5 tumors per mouse at 200 days). Interestingly, tumors (>2 mm) from the Rapa-fed group showed a reduction in UV-signature p53 mutations (39% vs 90%) in favor of mutations from putative base oxidation. This shift in mutation spectrum was not essentially linked to the reduction in large tumors because it was absent in large tumors similarly reduced in number when feeding Rapa in combination with MMF, possibly owing to an antioxidant effect of MMF. Significantly fewer tumor cells were Vegf-positive in the Rapa-fed groups, but a correspondingly reduced expression of Hif1alpha target genes (Vegf, Ldha, Glut1, Pdk1) that would indicate altered glucose metabolism with increased oxidative stress was not found. Remarkably, we observed no effect of the immunosuppressants on UV-induced tumor onset, and with impaired tumor outgrowth Rapa could therefore strongly reduce skin carcinoma morbidity and mortality rates in organ transplant recipients.

Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Isolation and characterization of permanent cell lines from inner cell mass cells of bovine blastocysts.

Inner cell masses (ICM) from in vitro produced day 8 or 9 bovine blastocysts were isolated by immunosurgery and cultured under different conditions in order to establish which of two feeder cell types and culture media were most efficient in supporting attachment and outgrowth of the bovine ICM cells. The efficiency of attachment and outgrowth of the ICM cells could be markedly improved when STO feeder cells were used instead of bovine uterus epithelial cells, and by using charcoal-stripped serum instead of normal serum to supplement the culture medium. More than 20 stable cell lines were obtained. Some of these lines were examined by immunofluorescence for developmentally regulated markers. From these results we conclude that the cell lines resemble epithelial cells, rather than pluripotent ICM cells. The developmental potential of cells of one of the lines was tested in the nuclear transfer assay. The cell line could support the initial development of enucleated oocytes, but none of the reconstructed embryos passed the eight-cell block.

Stage-specific appearance of the mouse antigen TEC-3 in normal and nuclear transfer bovine embryos: re-expression after nuclear transfer.

Bovine embryos, recovered from the uterus in vivo or derived from in vitro matured and in vitro fertilized oocytes, were investigated for the presence of the developmentally regulated mouse antigen TEC-3 by indirect immunofluorescence. During preimplantation embryo development TEC-3 is expressed on bovine morulae and blastocysts. It is absent from unfertilized and fertilized oocytes, and from all stages before the 32-cell stage. The finding that TEC-3 is not expressed before the onset of embryonic transcription, which occurs at the eight-cell stage in the bovine, but only when the embryonic genome is active, makes it a potential marker for studying nuclear reprogramming after nuclear transfer. Nuclear transfer embryos were made by electrical fusion of blastomeres from morulae derived in vivo with enucleated metaphase II oocytes. Indirect immunofluorescence with the TEC-03 antibody showed that the TEC-3 antigen, present on blastomeres of the morula stage embryo, disappeared after fusion and was expressed again when the nuclear transfer embryos developed to the morula and blastocyst stage. These data suggest that the bovine embryonic nucleus may be able to revert to the equivalent of an earlier developmental stage when transferred to ooplasm, and is then capable of following the normal developmental program.