James E. Strickland

Critical Aspects of Initiation, Promotion, and Progression in Multistage Epidermal Carcinogenesis

Abstract Carcinogenesis in mouse skin can be divided into three distinct stages: initiation, promotion, and progression (malignant conversion). Initiation, induced by a single exposure to a genotoxic carcinogen, can result from a mutation in a single critical gene (e.g., rasHa), apparently in only a few epidermal cells. The change is irreversible. Promotion, resulting in the development of numerous benign tumors (papillomas), is accomplished by the repeated application of a nonmutagenic tumor promoter. The effects of single applications of tumor promoters are reversible since papillomas do not develop after insufficient exposure of initiated skin to promoters or when the interval between individual promoter applications is increased sufficiently. The reversibility of promotion suggests an epigenetic mechanism. Promoter treatment provides an environment that allows the selective clonal expansion of foci of initiated cells. The conversion of squamous papillomas to carcinomas (termed progression or malignant conversion) occurs spontaneously at a low frequency. The rate of progression to malignancy can be significantly increased by treatment of papilloma-bearing mice with certain genotoxic agents. These progressor agents or converting agents are likely to act via a second genetic change in papillonias already bearing the initiating mutation. Progression in the skin is characterized by genetic changes that result in several distinct changes in the levels or activity of structural proteins, growth factors, and proteases. The mechanisms involved in progression are being studied in epidermal cell culture. In order to determine the in vivo phenotype of cultured cells, a grafting system was developed in which the cells were transferred from culture to a prepared skin bed in athymic mice. Introduction of an activated v-fos oncogene into initiated cells bearing an activated ras Ha gene produced cells with a carcinoma phenotype, of Initiation i.e., carcinomas formed when the cells were grafted as part of reconstituted skin. Grafted keratinocytes containing the ras Ha gene alone produced papillomas; with v-fos alone, normal skin formed when grafted. The ras Ha/fos carcinomas showed changes in differentiation markers characteristic of chemically induced carcinomas. A cell culture assay utilizing cells initiated by the introduction of an activated ras Ha oncogene was developed to study progression. After exposure of initiated cells to progressor agents under conditions in which the proliferation of the ras Ha-initiated cells was suppressed, proliferating foci developed, with a good correlation of activity in the assay with activity in the progression stage in vivo. The cell culture assay provides a quantitative model to study chemically induced neoplastic progression and may be useful to identify potential progressor agents.

Direct isolation of xenotropic retraviruses from the NIH swiss mouse uterus.

Abstract Xenotropic type C retraviruses were isolated from cell-free uterine extracts of normal adult NIH Swiss mice by direct inoculation of mink, cat, and human target cells. Successful isolation occurred in approximately one-half the susceptible target cell cultures inoculated with a given extract. These results demonstrate the presence of fully infectious xenotropic virus particles in the uterus of the normal NIH Swiss mouse.

Group specific antigen and RNA-directed DNA polymerase activity in normal baboon placenta.

Summary C-Type particles were observed in normal baboon placentas at various stages of gestation. This tissue was found to have interspecies specific antigens of the C-type RNA tumor viruses and RNA-directed DNA polymerase activity that could not be inhibited by antisera prepared against the previously recognized reverse transcriptases of several RNA tumor viruses. This study was funded in part by Contract NIH-NCI-E-71-2348 from the Special Virus Cancer Program, NCI; Grant RR00361 from the U.S. Public Health Service and WHO Grant Z2/181/27. The laboratory at the Southwest Foundation for Research and Education serves as the WHO Regional Reference Center for Simian Viruses.

Oncornaviral Protein Modulation in Mouse Uterine Tissue by Estrogen

Summary Treatment of ovariectomized NIH Swiss mice with estrogens elevated the level of the murine leukemia virus group specific protein and the activity of an RNA-di-rected DNA polymerase in the uterus. The extent that these markers were raised was dependent on the relative biological potency of the estrogen and on the time interval following treatment. Increases in the levels of both viral marker proteins were evident within 24 hr of treatment and were highest at 48 hr. Subsequently, viral protein levels declined to pretreatment levels. The authors acknowledge the technical assistance of Carl Reed, Carol Lipson, Leon Culler, Sara Myers, and Philip Errico. Antisera used in this study were prepared by Dr. R. Wilsnack, Huntington Research Center, Baltimore, MD.

Antigenic determinants shared by the DNA polymerases of reticuloendotheliosis virus and mammalian type C retroviruses

Abstract Antiserum to the DNA polymerase of spleen necrosis virus, a reticuloendotheliosis (RE) group virus, inhibited the polymerase activity of reverse transcriptases from two RE group virus isolates and from mammalian type C retroviruses of murine, feline, and primate origin, but did not inhibit reverse transcriptases of avian myeloblastosis virus (AMV) or the type B or type D mammalian retroviruses tested. Conversely, antiserum to DNA polymerase of a mammalian type C retrovirus, Rauscher murine leukemia virus, inhibited the polymerases of mammalian type C viruses and a RE group virus but was ineffective against AMV polymerase.

The In Vitro Analysis of Biochemical Changes Relevant to Skin Carcinogenesis

The phenotypic alterations produced in mouse skin cells during the multistage development of squamous cancer have been well documented. In normal skin, all proliferating cells are confined to the basal cell compartment where less than 10% of the cells are in S phase when pulse-labeled with DNA precursors. Two keratins, K5 (M r 60 000) and K14 (M r 55 000), are transcribed largely in basal cells, although the proteins persist in the upper layers (Roop et al. 1988). The commitment to differentiate is associated with the loss of proliferative potential, the commencement of suprabasal migration, and the expression of two suprabasal keratins, K1 (M r 67 000) and K10 (M r 59 000) in the first spinous cell layer (Roop et al. 1988). Proliferating cells do not express K1 or K10 in normal epidermis. As cells migrate into the granular cell layer, K1 and K10 transcripts diminish and the genes for filaggrin, a M r 27 000 interfilamentous matrix protein, and loricrin, a major component of the cornified envelope, are activated and the proteins synthesized (Mehrel et al. 1990; Roop et al. 1989).

Estradiol Effect on Type C Viral Gene Expression in the Uterus of the Ovariectomized Mouse

Summary Kinetic studies using hybridization with complementary DNAs synthesized from high molecular weight type C viral RNAs showed that virus-specific sequences were among the earliest nonribosomal RNAs to accumulate in the mouse uterus after estrogen treatment. Following hormone administration to ovariectomized animals, significant increases above the low level of viral-specific RNA continuously present in the uterus were observed as early as 4 hr, with maximum levels at 8–16 hr. Subsequently, there was a decrease in the relative concentration of viral RNA. However, levels were still higher than basal at 24 hr, the last time point examined. The increase in viral RNA occurred before a detectable increase in protein synthesis, suggesting that this viral-specific RNA was probably mRNA. We thank Thomas Paisley and Carl Reed for excellent technical assistance with this study and Jean McCammon and Audrey Brammer for preparing the manuscript.

Impaired Estrogen-Mediated Production of Type C Viral DNA Polymerase in Aged NIH Swiss Mouse Uteri

Summary Effects of estrogen administration were compared in young (2-5 months) and aged (2.0-2.5 years), ovariectomized NIH Swiss mice. Two murine leukemia virus proteins, p30 and RNA-directed DNA polymerase, were markedly elevated by estrogen in the uteri of young, ovariectomized mice, but behaved differently in aged animals. The polymerase was low or absent in the uteri of aged mice and showed no increase in response to estrogen. In contrast, p30 responded to estrogen in the aged animals much the same as in the younger mice. This implies that the production of these viral proteins is under separate control mechanisms. The authors gratefully acknowledge the excellent technical assistance of Glenn He-gamyer, Carl Reed, Cindy Lin, and Leon Culler. We thank Ruby Howard for histo-logical preparations, and Dr. Richard Olsen for histological evaluation of our tissue specimens.