Nam-ho Huh

Growth and Differentiation Properties of Normal and Transformed Human Keratinocytes in Organotypic Culture

The growth and differentiation of human normal keratinocytes and their transformed counterparts were examined in organotypic cultures in which the keratinocytes were grown at the air‐liquid interface on top of contracted collagen gel containing fibroblasts. We developed a modified culture procedure including the use of a mixed medium for keratinocytes and fibroblasts. Normal keratinocytes formed a three‐dimensional structure of epithelium that closely resembled the epidermis in vivo, consisting of basal, spinous, granular and cornified layers. Cells synthesizing DNA were located in the lowest basal layer facing the collagen gel. Expressions of proteins involved in epidermal differentiation were examined by immunohistochemical staining and compared with those in skin in vivo. In the organotypic culture, transglutaminase, involucrin and filaggrin were expressed, as in the epidermis in vitro, most prominently in the granular layer. Type IV collagen, a component of basement membrane, was expressed at the interface between the keratinocyte sheet and the contracted collagen gel. Keratinocytes transformed by simian virus 40 or human papilloma virus (HPV) exhibited a highly disorganized pattern of squamous differentiation. In particular, HPV‐transformed cells invaded the collagen gel. Organotypic culture is unique in that regulatory mechanisms of growth and differentiation of keratinocytes can be investigated under conditions mimicking those in vivo.

Why are human cells resistant to malignant cell transformation in vitro

Transformation of human cells, both induced and spontaneous, is an extremely rare event, whereas rodent cells are relatively easily transformed when treated with a single carcinogenic agent. The present review addresses the question of why human cells are resistant to malignant transformation in vitro. To facilitate understanding of the problem, the process of transformation is divided operationally into two phases, i.e. phase I, immortalization; and phase II, malignant transformation. In human cells, one‐phase transformation, i.e., the consecutive occurrence of phases I and II due to the action of a single carcinogenic agent, is observed only rarely. Once human cells are immortalized, however, malignant transformation by chemical carcinogens or oncogenes proceeds, suggesting that for human cells, phase I immortalization is a prerequisite for such transformation to take place. To date, about 20 papers have been published describing protocols for the two‐phase transformation of a variety of human epithelial cells and fibroblasts. In most experiments, SV40, human papilloma viruses and their transforming genes are utilized for induction of phase I (immortalization) followed by the use of chemical carcinogens or activated oncogenes for induction of phase II (malignant transformation). Possible mechanisms that would render human cells refractory to transformation are discussed below.

DMSO induces apoptosis in SV40-transformed human keratinocytes, but not in normal keratinocytes

We found that dimethyl-sulfoxide (DMSO) at concentrations of 2.5% induced apoptosis in SV40-immortalized human keratinocytes, while normal keratinocytes were arrested at the boundary of G1/S phase under the same conditions. DMSO-induced apoptosis in SV-40 immortalized keratinocytes was not associated with change in phosphorylated state of the retinoblastoma susceptibility gene. When SV40-immortalized cells were treated with 2.5% DMSO, dissociation of the complex was observed by immunoblotting of SV40 T antigen from immunoprecipitated p53 protein fraction.

Formation and Enzymatic Repair of Specific Reaction Products of Alkylating N-Nitroso Carcinogens in Cellular DNA: Relevance to Malignant Transformation

Structural modifications of genomic DNA, with the ensuing qualitative and quantitative alterations in the patterns of gene expression in the respective target cells and their progeny, appear to be key elements in the process of malignant transformation and tumorigenesis caused by cellular interactions with chemical carcinogens, radiation, or viruses. It is likely that the specific DNA sequence alterations (mutations) recently observed in genes associated with the tumorigenic conversion of various types of human and animal cells, may, at least in part, be traced back to the formation of specific adducts in genomic DNA by DNA-reactive agents from our environment.1,2 Of particular relevance with respect to the probability (“risk”) of carcinogen-induced malignant conversion of individual cells are (i) their capacity for enzymatic bioactivation of “precarcinogens” to their reactive derivatives (if such derivatives are not generated without enzymatic catalysis, i.e., by spontaneous decomposition of the parent compound),3 and (ii) their capacity for repair of damaged DNA in critical gene sequences via the action of specific repair enzymes.4 It goes without saying that the varying capacity of cells for enzymatic repair of specific DNA adducts also plays an important role with regard to cancer therapy, inasmuch as repair proficiency increases the resistance of cells towards the cytotoxic effects of DNA-reactive therapeutic agents.5 The present chapter focuses on N-nitroso compounds as an example of a large class of DNA-reactive alkylating carcinogens, using as a model a particularly potent carcinogen and mutagen, N-ethyl-N-nitrosourea (EtNU).4,6,7 Some aspects of the formation and distribution of the different alkylation products in genomic DNA will be discussed as well as the current methodology for their sensitive detection by monoclonal antibodies. Moreover, we will briefly describe results of experiments aiming at an evaluation of the relative importance of the selective enzymatic repair of one of these DNA alkylation products, O6-ethylguanine (O6-EtGua), as a determinant reducing the cellular risk of malignant transformation.