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.

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

Caspases are intracellular proteases that function as initiators and effectors of apoptosis. The kinase Akt and p21-Ras, an Akt activator, induced phosphorylation of pro-caspase-9 (pro-Casp9) in cells. Cytochrome c-induced proteolytic processing of pro-Casp9 was defective in cytosolic extracts from cells expressing either active Ras or Akt. Akt phosphorylated recombinant Casp9 in vitro on serine-196 and inhibited its protease activity. Mutant pro-Casp9(Ser196Ala) was resistant to Akt-mediated phosphorylation and inhibition in vitro and in cells, resulting in Akt-resistant induction of apoptosis. Thus, caspases can be directly regulated by protein phosphorylation.

https://science.sciencemag.org/content/sci/282/5392/1318.full.pdf

Regulation of Cell Death Protease Caspase-9 by Phosphorylation

For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

https://cancerres.aacrjournals.org/content/canres/48/3/589.full.pdf

Feasibility of Drug Screening with Panels of Human Tumor Cell Lines Using a Microculture Tetrazolium Assay

During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes1,2. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants3,4,5, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization6, the selection of rare, spontaneously arising immortalized cells7,8,9,10, or the use of an entire viral genome11. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT)12 in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.

Creation of human tumour cells with defined genetic elements

Platelet-derived growth factor (PDGF) is a major mitogen for connective tissue cells and certain other cell types. It is a dimeric molecule consisting of disulfide-bonded, structurally similar A- and B-polypeptide chains, which combine to homo- and heterodimers. The PDGF isoforms exert their cellular effects by binding to and activating two structurally related protein tyrosine kinase receptors, denoted the alpha-receptor and the beta-receptor. Activation of PDGF receptors leads to stimulation of cell growth, but also to changes in cell shape and motility; PDGF induces reorganization of the actin filament system and stimulates chemotaxis, i.e., a directed cell movement toward a gradient of PDGF. In vivo, PDGF has important roles during the embryonic development as well as during wound healing. Moreover, overactivity of PDGF has been implicated in several pathological conditions. The sis oncogene of simian sarcoma virus (SSV) is related to the B-chain of PDGF, and SSV transformation involves autocrine stimulation by a PDGF-like molecule. Similarly, overproduction of PDGF may be involved in autocrine and paracrine growth stimulation of human tumors. Overactivity of PDGF has, in addition, been implicated in nonmalignant conditions characterized by an increased cell proliferation, such as atherosclerosis and fibrotic conditions. This review discusses structural and functional properties of PDGF and PDGF receptors, the mechanism whereby PDGF exerts its cellular effects, and the role of PDGF in normal and diseased tissues.

https://www.physiology.org/doi/pdf/10.1152/physrev.1999.79.4.1283

Mechanism of Action and In Vivo Role of Platelet-Derived Growth Factor

In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors.

A tumor cell line was derived from the fibrosarcoma of a 35-year-old Caucasian man who died without having received chemotherapy or radiotherapy. The in vitro growth properties and transplantability into antithymocytic sera treated mice were characteristic of these malignant cells. An aberrant karyology with marker chromosomes was present. No virus particles were detected. Cancer 33:1027-1033, 1974. ERMANENT CELL LINES FROM ANIMAL TISSUES P were established as early as 1948.23 Most of these were originated from malignant tissues. Although a number of human cell lines have been established in vitro, very few fibrosarcoma cell lines have been described. As far as we are aware, there are reports of approximately eight cell lines derived from osteogenic sarcomas,1,4,Q,10,13,~1 two on rhabdomyosarcoma,12)26 three on liposarcoma,1.9.10 and one each on giant cell sarcomas,l Kaposi's sarcoma,* neurogenic sarcoma,26 synovioma,lO and fibrosarcoma.l The rest of the reported cell lines are all carcinomas. Only a few of these sarcoma cell lines have been fully studied and characterized. In this paper we describe the properties of a tumor cell line (HT1080) derived from a fibrosarcoma of a human male.

Characterization of a newly derived human sarcoma cell line (ht-1080)

Recent investigations have begun to dissect the number and nature of genetic alterations associated with cancer cells. In the present study, primary human epidermal keratinocytes acquired indefinite life-span in culture but did not undergo malignant conversion in response to infection with a hybrid of adenovirus 12 and simian virus 40. Addition of Kirsten murine sarcoma virus, which contains a K-ras oncogene, to these cells induced morphological alterations associated with the acquisition of neoplastic properties. These findings demonstrate the malignant transformation of human primary epithelial cells in culture and support a multiple-step process for neoplastic conversion.

https://science.sciencemag.org/content/sci/227/4691/1250.full.pdf

Neoplastic Transformation of Human Epidermal Keratinocytes by AD12-SV40 and Kirsten Sarcoma Viruses

An infectious C-type feline sarcoma virus (FSV), isolated from a naturally occurring fibrosarcoma of a domestic house cat, produces fibrosarcomas in kittens and puppies and induces cell transformation in feline embryo fibroblasts. Prenatal inoculation of cat and dog foetuses is particularly effective in tumour transmission. Postnatal dogs bearing FSV induced fibrosarcomas provide antisera suitable for detecting the feline C-type RNA tumour virus antigens in the complement fixation test.

Experimental Transmission of Feline Fibrosarcoma to Cats and Dogs

Two newly established human bladder carcinoma cell lines, designated HT-1197 and HT-1376, were characterized. Cells of both cultures exhibited fine structural microvilli and tonofibrils indicative of their epithelial origin. In addition, desmosomes were also present in HT-1197. Marker chromosomes present in HT-1197 and HT-1376 distinguished these from each other and from other known human tumor cell lines. Both cultures grew in soft agar, induced fibrinolytic activity, and were tumorigenic in mice and hamsters. No type C or other virus expression was detected in these cell lines nor in other human urothelial tumors tested.

Paul Arnstein

Papers

In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors.

Characterization of a newly derived human sarcoma cell line (ht-1080)

Neoplastic Transformation of Human Epidermal Keratinocytes by AD12-SV40 and Kirsten Sarcoma Viruses

Experimental Transmission of Feline Fibrosarcoma to Cats and Dogs

Human bladder carcinoma: characterization of two new tumor cell lines and search for tumor viruses.