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.

Changes in keratinocyte maturation during wound healing.

• With ion capture cytochemistry, we previously demonstrated the distribution of calcium ions in murine epidermis, a pattern consistent with a role for this ion in the regulation of epidermal differentiation. Because of the known proliferation and differentiation defects in psoriasis, we compared the calcium distribution of involved vs uninvolved psoriatic lesions and normal human epidermis. Whereas normal human and uninvolved psoriatic epidermis revealed increased calcium-containing precipitates in the uppermost stratum granulosum, in contrast the basal layer of psoriatic lesions contained less extracellular calcium, a condition that favored enhanced proliferation. Moreover, all psoriatic suprabasal cell layers displayed heavier than normal concentrations of calcium, indicating loss of the normal calcium gradient that programs terminal differentiation. This abnormal profile may account for the differentiation defects (eg, parakeratosis) that occur in psoriasis. Finally, psoriatic lesions displayed retained ionic Ca in intercellular domains of the upper stratum granulosum with absence of normal intercellular bilayers, findings that may underlie the abnormal desquamation and permeability barrier in psoriasis. ( Arch Dermatol. 1991;127:57-63)

Ultrastructural localization of calcium in psoriatic and normal human epidermis.

In vitro transformation of human epithelial cells.

Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains. In this paper, the expression and function of integrins in psoriatic keratinocytes were examined, both in vivo and in vitro. We found that: (a) in psoriatic keratinocytes the integrin heterodimers alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 have lost their polarized distribution on the plasma membrane; (b) the role of these integrins in mediating keratinocyte adhesion in vitro is altered; (c) psoriatic keratinocytes form focal contacts containing both beta 1 and beta 4 integrins. In normal adult keratinocytes the alpha 5 beta 1 fibronectin receptor is poorly expressed and diffusely distributed on the basal keratinocyte plasma membrane and is not organized in defined adhesive structures. In contrast, psoriatic keratinocytes show a clear fibronectin receptor staining in vivo, and organize alpha 5 beta 1 in typical focal contacts in vitro without any obvious increase of its expression and synthesis. These multiple alterations of integrins are also present in uninvolved keratinocytes from psoriatic patients, suggesting a key role for altered integrin-mediated adhesion in the pathogenesis of this disease.

/pdf/expression-topography-and-function-of-integrin-receptors-are-58slquqxo4.pdf

Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.

SUMMARY

We compared the maturation pathway of normal and psoriatic epidermis using three different markers: (I) Involucrin, which is normally detected in the stratum granulosum in normal skin, was detected in all but the basal layer of involved psoriatic skin; (2) an antigen, recognized by the murine monoclonal antibody Ψ3, was present in all but the basal layer of involved psoriatic skin but was absent from uninvolved and normal skin; (3) fibronectin, which normally localizes in the dermis and the epidermal-dermal junction, was also detected intra- and extracellularly in the psoriatic epidermis. These results indicate that the alterations in keratinocyte maturation found in psoriasis do not arise from a truncation of the normal maturation pathway but rather reflect the onset of an abnormal pathway of differentiation characterized by the expression of Ψ3 antigen and fibronectin and the premature appearance of involucrin.

Abnormal maturation pathway of keratinocytes in psoriatic skin.

Normal human keratinocytes are able to stratify, form cornified squames, and terminally differentiate in tissue culture. These properties are frequently impaired by malignant transformation. In the present paper, we show that, in addition, transformation by SV40 results in the coordinate reexpression of a whole set of fetal characters. Moreover, a comparison of two SV40-transformed human keratinocyte cell lines, one still showing a certain degree of stratification and terminal differentiation (HE-SV) and the other almost completely unable to differentiate (SVK14), suggests that the impairment of differentiation and the intensity of reexpression of fetal markers are correlated. Particularly, a set of three keratin polypeptides, absent in adult stratified epithelia but normally found in the nonstratified fetal epidermis, is present in much larger amounts in SVK14 cells than in HE-SV cells. On the other hand, the inability of SVK14 cells, in contrast to HE-SV cells, to form cornified envelopes seems to be due to the inability of those cells to accumulate involucrin.

/pdf/reexpression-of-fetal-characters-in-simian-virus-40-36zjgu8kj1.pdf

Reexpression of fetal characters in simian virus 40-transformed human keratinocytes.

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Abnormal maturation pathway of keratinocytes in psoriatic skin.

Reexpression of fetal characters in simian virus 40-transformed human keratinocytes.