Rajiv R. Mohan

Apoptosis is an early event during phthalocyanine photodynamic therapy-induced ablation of chemically induced squamous papillomas in mouse skin.

Abstract— Photodynamic therapy (PDT) is a promising new modality to treat malignant neoplasms including superficial skin cancers. In our search for an ideal photosensitizer for PDT, Pc 4, a silicon phthalocyanine, has shown promising results both in in vitro assays and in implanted tumors. In this study we assessed the efficacy of Pc 4 PDT in the ablation of murine skin tumors; and the evidence for apoptosis during tumor ablation was also obtained. The Pc 4 was administered through tail vein injection to SENCAR mice bearing chemically induced squamous papillomas, and 24 h later the lesions were illuminated with an argon ion‐pumped dye laser tuned at 675 nm for a total light dose of 135 J/cm2. Within 72‐96 h, almost complete tumor shrinkage occurred; no tumor regrowth was observed up to 90 days post‐PDT. As evident by nucleosome‐size DNA fragmentation, appearance of apoptotic bodies in hematoxylin and eosin staining and direct immunoperoxidase detection of digoxigenin‐labeled genomic DNA in sections, apoptosis was clearly evident 6 h post‐PDT at which time tumor shrinkage was less than 30%. The apoptotic bodies, as evident by the condensation of chromatin material around the periphery of the nucleus and increased vacuolization of the cytoplasm, were also observed in electron microscopic studies of the tumor tissues following Pc 4 PDT. The extent of apoptosis was greater at 15 h than at 6 and 10 h post‐PDT. Taken together, our results clearly show that Pc 4 may be an effective photosensitizer for PDT of nonmelanoma skin cancer, and that apoptosis is an early event during this process.

Protection against malignant conversion in SENCAR mouse skin by all Trans retinoic acid: Inhibition of the ras p21‐processing enzyme farnesyltransferase and Ha‐ras p21 membrane localization

Many studies have shown that all trans retinoic acid (RA) exhibits significant protective effects against mouse skin tumor promotion and spontaneous as well as enhanced malignant conversion. In a recently completed study, we showed that under treatments in which papillomas on SENCAR mouse skin are induced at low and high probabilities to convert to malignant carcinomas, RA affords significant protection against both tumor promotion and subsequent malignant conversion. More than 95% of these mouse skin papillomas and carcinomas have been shown to contain point mutation at the 61 codon of Ha‐ras oncogene. The ras oncogene encodes a p21 protein that, in its mutated form, transforms mammalian cells only when p21 is at the inner surface of the plasma membrane, by a series of enzymatic reactions in which the initial step is catalyzed by farnesyltransferase (FTase). In this study, we assessed whether the protective effect of RA against malignant conversion involves the inhibition of ras p21 processing in those tumors that contain the activated ras oncogene. The FTase activity and the levels of cytosolic and membrane‐bound Ha‐ras p21 were determined in all papillomas and carcinomas obtained from acetone‐ or RA‐treated animals. No matter how the data were analyzed and what comparisons were considered, in all the protocols used, compared with controls, papillomas and carcinomas obtained from RA‐treated groups showed significantly decreased (P < 0.01–0.001) FTase activity. Furthermore, the tissue samples from RA‐treated groups in different protocols also showed significantly diminished membrane localization of Ha‐ras p21, with a concomitant increase in cytosolic Ha‐ras p21 levels. The analysis of these data also showed that in all the protocols used, the increased FTase activity and membrane localization of Ha‐ras p21 were associated with the induction of papillomas and their subsequent malignant conversion to squamous cell carcinomas. Taken together, these results indicate a strong correlation between the inhibition of ras p21 farnesylation because of a decrease in FTase activity by RA and its protective effect against malignant conversion of papillomas to carcinomas. Based on the results of this study, it is tempting to suggest that clinical trials evaluating the preventive or therapeutic potential of retinoids may be directed more toward those clinical malignancies that are known to contain the activated ras oncogene.

Mutations in ras oncogenes: rare events in ultraviolet B radiation-induced mouse skin tumorigenesis.

The activation of ras proto‐oncogenes by point mutation in a broad spectrum of clinical malignancies and experimentally induced tumors suggests their critical role in cancer induction. To determine whether the activation of ras proto‐oncogenes by point mutation also contributes to ultraviolet B radiation (UVB)‐induced skin tumorigenesis and whether this event is responsible for the different tumorigenic potentials of UVB radiation in different mouse strains, we analyzed the skin tumors induced by UVB in SKH‐1 hairless and C3H mice for specific mutations in the Ha‐, Ki‐, and N‐ras oncogenes. With the same UVB irradiation protocol, the latency period for tumor appearance was longer in C3H mice than in SKH‐1 hairless mice. In addition, tumor incidence and multiplicity were also significantly higher (P < 0.001, χ2 and Wilcoxon rank sum tests) in SKH‐1 hairless mice compared with C3H mice. None of the 30 skin tumor specimens (15 from each mouse strain) analyzed by polymerase chain reaction (PCR) amplification of specific codons followed by dot‐blot hybridization with specific probes contained mutation in codons 13 of Ha‐ras; 12, 13, and 61 of Ki‐ras; or 12 and 13 of N‐ras. However, three of the 15 tumors in SKH‐1 hairless mice showed either a G35 → A or G35 → T transition at second position of Ha‐ras codon 12. Interestingly, one of these tumors (with a G35 → A transition) also harbored an A182 → G mutation at second position of Ha‐ras codon 61. None of the tumors from C3H mice showed mutations in codons 12 or 61 of the Ha‐ras oncogene. With regard to codon 61 of the N‐ras oncogene, six tumors from SKH‐1 hairless mice and 10 tumors from C3H mice showed an A183 → T transversion. While G35 → A or G35 → T transition detected by PCR and dot‐blot hybridization was confirmed by sequencing, the mutations identified similarly at codon 61 in either the Ha‐ or N‐ras oncogenes could not be verified by sequencing of PCR‐amplified products subcloned into plasmid vectors. With the exception of the low incidence of Ha‐ras oncogene mutations at codon 12 in SKH‐1 hairless mouse skin tumors induced by UVB, the striking absence of mutations in the Ha‐, Ki‐, and N‐ras oncogenes in UVB‐induced mouse skin tumors suggests that ras oncogene mutations are rare and thus are not an initiating event in photocarcinogenesis.