In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.

/pdf/normal-keratinization-in-a-spontaneously-immortalized-5753ggq1uq.pdf

Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.

https://www.cell.com/cell/pdf/0092-8674(92)90189-J.pdf

Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Two sublines of a breast epithelial cell culture, MCF-10, derived from human fibrocystic mammary tissue exhibit immortality after extended cultivation in low calcium concentrations (0.03-0.06 mM) and floating transfers in low calcium (MCF-10F), or by trypsin-Versene passages in the customary (normal) calcium levels, 1.05 mM (MCF-10A). Both sublines have been maintained as separate entities after 2.3 years (849 days) in vitro and at present have been in culture for longer than 4 years. MCF-10 has the characteristics of normal breast epithelium by the following criteria: (a) lack of tumorigenicity in nude mice; (b) three-dimensional growth in collagen; (c) growth in culture that is controlled by hormones and growth factors; (d) lack of anchorage-independent growth; and (e) dome formation in confluent cultures. Cytogenetic analysis prior to immortalization showed normal diploid cells; although later passages showed minimal rearrangement and near-diploidy, the immortal cells were not karyotypically normal. The emergence of an immortal culture in normal calcium media was not an inherent characteristic of the original tissue from which MCF-10 was derived since reactivated cryo-preserved cells from cultures grown for 0.3 and 1.2 years in low calcium were incapable of sustained growth in normal calcium.

https://cancerres.aacrjournals.org/content/canres/50/18/6075.full.pdf

Isolation and Characterization of a Spontaneously Immortalized Human Breast Epithelial Cell Line, MCF-10

http://sun-lab.med.nyu.edu/files_private/sun-lab/attachments/1989.Cell.Cotsarelis-s.pdf

Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells.

Exposure of mouse epidermal cells in culture to 12- O -tetradecanoyl-phorbol-13-acetate (TPA) results in an initial inhibition of DNA synthesis for 24 hr followed by a 5- to 10-fold stimulation at 72 to 96 hr. A corresponding increase in mitotic rate also occurs at 72 to 96 hr. These responses occur when TPA is continuously present in the medium or if the exposure is as short as 1 hr, but the degree of stimulation was dependent on dose and duration of exposure. Sensitivity to TPA varied with the length of time the cells were in culture prior to treatment. TPA treatment also produced an alteration in morphology from clearly epithelial to a more fibroblastic type. These biochemical and morphological effects did not occur after treatment of epidermal cells with either phorbol-13,20-diacetate or phorbol. Primary dermal fibroblasts in culture did not respond to TPA in this manner, but a line of cultured liver epithelial cells was slightly stimulated by the promoter. This sytem appears to be a sensitive in vitro model for detecting the hyperplasia-inducing effects of phorbol esters and should be useful for mechanistic studies and bioassay.

Stimulated DNA synthesis in mouse epidermal cell cultures treated with 12-O-tetradecanoyl-phorbol-13-acetate.

Methods to culture cells from papillomas induced by an initiation-promotion protocol in SENCAR mice were developed, and the resultant cell lines have been characterized Using Eagle's medium with 005 mM Ca2+ conditioned by dermal fibroblasts and supplemented with 1 ng/ml epidermal growth factor (EGF) in culture dishes coated with collagen and fibronectin, six cell lines (PA, PB, PC, PD, PE and PF) were established from separate pools of papillomas When tested for tumorigenicity in nude mice by injection of a cell suspension or implantation of cells growing on a plastic liner, two of the lines (PC and PF) produced no tumors at any passage In contrast, cells of the lines PA and PE produced highly differentiated squamous cell carcinomas from the earliest passage tested The results with PB and PD were variable on tumorigenicity testing with some passages positive and others negative When tested for responsiveness to Ca2+ (greater than 01 mM) as a differentiation stimulus, all lines responded In the higher Ca2+ medium there was a 50-95% decrease in colony-forming efficiency, a slight decrease in [3H]thymidine incorporation (except for PA) and an increase in the number of cornified cells (except for early passage PF) Epidermal transglutaminase activity, a marker for terminal differentiation, was increased in the presence of medium with Ca2+ greater than 01 mM However, unlike normal cells, only a fraction of the cells from each of the papilloma-derived cell lines terminally differentiated in response to Ca2+ while the remaining cells continued to proliferate, although at a slower rate Responsiveness to phorbol ester tumor promoters was also examined in papilloma cell lines 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment increased colony forming efficiency, DNA synthesis and colony size in all lines in medium with either 005 mM Ca2+ or 12 mM Ca2+ TPA treatment also increased ornithine decarboxylase activity in all lines, even at the higher Ca2+ concentration, although normal keratinocytes respond only when grown in medium with low Ca2+ TPA treatment caused only a slight increase in the number of cornified cells and no increase in epidermal transglutaminase activity in papilloma cells while it causes 10-fold or greater increases in these differentiation markers in normal keratinocytes Thus papilloma cells appear to differ from normal keratinocytes in their ability to maintain a proliferating population under conditions favoring terminal differentiation, their consistent proliferative response to phorbol esters under these same conditions, and their reduced sensitivity to phorbol ester-induced terminal differentiation(ABSTRACT TRUNCATED AT 400 WORDS)

Delores Michael

Papers

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Stimulated DNA synthesis in mouse epidermal cell cultures treated with 12-O-tetradecanoyl-phorbol-13-acetate.

Cultivation and characterization of cells derived from mouse skin papillomas induced by an initiation-promotion protocol.

Purified Epidermal Pentapeptide Inhibits Proliferation and Enhances Terminal Differentiation in Cultured Mouse Epidermal Cells

Response of carcinogen-altered mouse epidermal cells to phorbol ester tumor promoters and calcium