Mark L. Steinberg

Isolation and characterization of a spontaneously arising long-lived line of human keratinocytes (NM1)

SummaryThe long-lived keratinocyte line, NM1, was isolated from the epidermis of a pool of foreskins obtained from apprently, normal neonates at the time of circumcision. Cultures were initiated in Dulbecco’s minimal essential medium containing 20% fetal bovine serum, 0.4 μg/ml hydrocortisone, 10−9M cholera, toxin, and 10 ng/ml epidermal growth factor using mitomycin C-treated 3T3 cells as a feeder layer. Unlike normal keratinocytes which survive for only 150 generations these cells have been in culture for more than a year and have been carried for more than 400 doublings. The cells seem to follow a pathway, of growth and differentiation that is very similar to normal keratinocytes. Cytokeratin fibrils, intercellular attachments, and cornified envelopes were observed. The keratin polypeptides isolated from the NM 1 cells were similar to those previously described in normal cultured, cells; the presence of profilaggrin and involucrin was demonstrated by sodium dodecyl sulfate electrophoresis and immunoblotting with monoclonal antibodies specific to these proteins. The NM 1 cells showed a reduced dependency on 3T3 feeder cells but did not form tumors when placed into athymic nude mice. Screening of the cells for SV40, BK, HPV 16, and HPV 18 viruses was negative. The NM1 cells showed trisomy of chromosome 8. The long-lived nature of these cells makes them a valuable model for studying growth and differentiation of kerationocytes.

The effect of different solvents on the ATP/ADP content and growth properties of HeLa cells.

Testing of the effects of xenobiotics in cultured cells often requires the use of organic solvents to effect suspension of the test agents in cell culture media. However, the toxic effects of the solvents themselves may introduce artifacts, which obscure interpretation of the experimental results. In this article, the toxicity of different solvents commonly used for solvation of a variety of xenobiotic agents was studied. We show that ethanol, acetone, isooctane, methanol, and hexane were considerably less toxic than the more commonly used solvent, DMSO, when ATP content and growth rates of HeLa cells exposed to these solvents was measured.

Simian virus 40-induced chromosome changes in human epidermal cultures

Abstract Chromosomal aberrations in human epidermal keratinocytes are described for the first time; the cells were transformed in vitro by the oncogenic virus, SV40 (Simian virus 40). The majority of metaphases exhibited hypotetraploid chromosome numbers and configurations indicative of frequent chromosomal breakage and fusion(s). The abnormalities were associated with segregational errors during various stages of mitosis; these errors were about 30 times as frequent in the transformed cells as in the uninfected keratinocytes. The pattern of karyotypic changes induced by the SV40 virus is similar to that previously observed in infected human fibroblasts, indicating that the alterations are viral-driven rather than cel-determined.

Re-expression of differentiated properties in SV40-lnfected human epidermal keratinocytes induced by 5-azacytidine☆

Infection of human epidermal keratinocytes by the oncogenic virus SV40 leads to progressive inhibition of the normal differentiation process in vitro. Treatment of infected cells with 5-azacytidine (5-aza-CR) over a 24-h period produced a striking enlargement and pronounced flattening of cells within 5-7 days following removal of the agent. This morphological change was accompanied by a several-fold increase in the number of cells staining positively for the cell envelope precursor protein, involucrin, and in the exfoliation of cornified envelope bearing cells from the monolayer. The drug-treated cultures at high passage levels were stained by immunofluorescence using monoclonal antibodies to keratin classes associated with different epidermal layers. These experiments revealed that 5-aza-CR caused the re-expression of two keratin classes (suprabasal and stratum corneum-associated), whose synthesis had been suppressed during the transformation process. 5-Aza-CR also brought about re-expression of 58 and 56 kD keratin markers of epithelial keratinization and stratification, as well as of 40 and 49-52 kD keratin markers of viral transformation. However, the responsiveness to the drug was gradually lost over time following infection.

Patterns of Persistent DNA Damage Associated with Sun Exposure and the Glutathione S-transferase M1 Genotype in Melanoma Patients

Solar radiation can lead to changes affecting DNA metabolism resulting in loss of DNA integrity. Skin specimens obtained from melanoma patients treated at the Memorial Sloan‐Kettering Cancer Center were used to study patterns of DNA fragmentation using the comet assay and levels of deletions in mitochondrial DNA (mtDNA) using real‐time PCR. Skin specimens were classified according to the glutathione S‐transferase M1 (GSTM1) genotype (either wild type [WT] or null) and patient sunburn history. GSTM1 null individuals with a sunburn history showed increased levels of both DNA fragmentation by comet assays and mtDNA deletions relative to GSTM1 WT patients with little or no sunburn history. Microarray analyses identified a number of genes whose expression was upregulated ≥5‐fold in cells from GSTM1‐null patients or from those reporting histories of sunburn. These genes encoded small molecule transporters, various growth factor/chemokine receptors, transcription factors and tumor suppressors. Of 17 genes directly involved in DNA repair, three DNA ligases were highly upregulated while the RAD23 UV excision repair gene and the Growth Arrest and DNA Damage gene (GADD45) were downregulated. These findings support the idea that exposure to solar radiation early in life may induce long‐term cellular changes that lead to persistent DNA damage and altered patterns of gene expression.

Fusion-induced differentiation of SV40-infected human keratinocytes☆

Abstract Cells in cultures of human keratinocytes infected by the oncogenic virus, SV40, were fused by exposure to polyethylene glycol (PEG). Fusion induced differentiation of the infected cells within 1–2 days, as measured by staining with orange G/acid fuchsin and by the production of cornified envelopes. The extent of induced differentiation was found to be correlated with the degree of multinucleation. The inductive effect of fusion was manifest during the early serial passages post-infection, but was eventually lost as the keratinocytes became increasingly transformed over time.

Synthesis of epidermolysis bullosa acquisita antigen by simian virus 40-transformed human keratinocytes

SummaryThe synthesis of epidermolysis bullosa acquisita (EBA) antigen in simian virus 40 (SV40)-transformed human epidermal keratinocytes was studied. Indirect immunofluorescent staining of SV40-transformed keratinocytes employing a serum sample from an EBA patient as a source of antibodies decorated EBA antigen as a perinuclear granular fluorescence. This staining pattern was similar to that of nontransformed epidermal keratinocytes grown in a low Ca2+ medium. In contrast, stratified primary cultures of keratinocytes stained after growth in a high Ca2+ medium showed only small amounts of the antigen localized in the substrate-attached basal cells. To demonstrate biosynthesis of the EBA antigen by SB40-transformed keratinocytes, cells were metabolically labeled with 14C-amino acids and the cell lysates were immunoprecipitated with EBA antiserum. Analysis of immunoprecipitates by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography revealed that the EBA serum precipitated a protein with an apparent molecular weight of 290 kD from extracts of these cells. These results indicate that SV40 induces the synthesis of the 290 kD EBA antigen. Expression of this antigen may be a general feature of nonstratifying, proliferating epidermal cells.

Proliferating Cell Nuclear Antigen/Cyclin in Cultured Human Keratinocytes

Expression of proliferating cell nuclear antigen (PCNA)/cyclin in cultured human keratinocytes was studied using an antibody from an SLE patient as the reagent. By indirect immunofluorescence staining, SV40‐transformed human keratinocytes expressed PCNA/cyclin in 40~45% of the cells as a nuclear granular fluorescence. After synchronization of these cells, their nuclear distribution pattern during the S phase was sequential and showed a clear correlation with DNA synthesis. Primary cultured keratinocytes grown in high Ca# medium expressed PCNA/cyclin in 10~15% of the cells with a similar staining pattern. These positively stained cells were confined to the basal and immediate suprabasal layers of the stratified culture sheet. The keratinocytes disaggregated by trypsin were separated according to cell size through a screen of Nitex monofilament cloth. The cells smaller than 15 µm in diameter synthesized abundant PCNA/cyclin, while the larger cells expressed very low levels. These results indicate that the expression of PCNA/cyclin correlates with DNA synthesis in cultured keratinocytes, but is not associated with their differentiation process.

Induction of Cyclin D1 by Arsenite and UVB-irradiation in Human Keratinocytes

Arsenic is an environmental pollutant with carcinogenic properties that is found in many regions of the world but that poses a health risk primarily in economically disadvantaged areas. In these areas, arsenic ingestion affects various tissues, especially skin in which it acts as a comutagen with the ultraviolet component of solar radiation. Both epidemiological and experimental evidence indicates that arsenic and ultraviolet radiation act on signaling pathways that effect the expression of cyclin D1. We have previously employed an in vitro model system of human epidermal keratinocytes to study the effects of submicromolar concentrations of sodium arsenite on cyclin D1 expression. Here, we employed this system to demonstrate concordant cyclin D1-related induction profiles of ultraviolet B radiation and arsenite using cDNA microarray analysis. We also show that both of these agents act epigenetically to bring about demethylation of the cyclin D1 promoter.

Spectrum of mitochondrial DNA deletions within the common deletion region induced by low levels of UVB irradiation of human keratinocytes in vitro

We show that a single low-dose exposure of human epidermal keratinocytes (NHEK) to an FS20 light source in vitro can induce the formation of mitochondrial DNA deletions in a PCR detection assay. We used primer sets specifically designed to exclude amplification of segments containing the common deletion, but which could detect possibly lower abundance deletions generated within the same region of the mitochondrial genome. We characterized eight novel deletions of which six were generated from cut sites within, or adjacent to, short direct repeats. Two deletions involved cut sites in inverted tetrameric repeats; one of these also involved an insertion.