Sustained nontumorigenic phenotype correlates with a largely stable chromosome content during long-term culture of the human keratinocyte line HaCaT

Abstract

Altered growth and differentiation and a highly abnormal karyotype are generally believed to be indicators for tumorigenic conversion of human cells. Inactivation of TP53 is supposedly one possible mechanism for accelerated genetic aberrations via reduced control of the genetic integrity. To examine the significance of this functional relationship, we investigated the long‐term development of the spontaneously immortalized human skin keratinocyte line HaCaT, carrying UV‐specific mutations in both alleles of the TP53 tumor suppressor gene. During >300 passages, proliferation, clonogenicity, and serum‐independent growth potential increased. In addition, HaCaT cells gained anchorage independence and at late passages showed reduced differentiation. Karyotypic analysis up to passage 225 revealed a high frequency of translocations and deletions, with a particular increase during passages 30 and 50. Nevertheless, the HaCaT cells remained nontumorigenic when injected subcutaneously, and noninvasive in surface transplants in nude mice. By comparative genomic hybridization, we confirmed the karyotypically identified phase of increased chromosomal aberrations between passages 30 and 50. However, before and thereafter, the CGH profiles of the individual chromosomes were largely unchanged, demonstrating that those translocations—also maintained in later passages—did not cause a gross chromosomal imbalance. Thus, our data suggest that multiple changes often correlated with a “transformed phenotype,” including extensive karyotypic alterations and mutational inactivation of TP53, are well compatible with a nontumorigenic phenotype of the HaCaT cells, and that preserved chromosomal balance may be crucial for this stability during long‐term propagation. Genes Chromosom. Cancer 19:201–214, 1997. © 1997 Wiley‐Liss, Inc.

UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes

“…Already a five-week-UVA-treatment is sufficient to induce tumorigenicity in three of the four populations. We previously showed that mere cultivation even for >6 years and >300 passages did not lead to tumorigenicity (Boukamp et al, 1997). Accordingly, mock-treated control cells remained nontumorigenic.…”

“…These cells represent an early stage of skin carcinogenesis because they harbor UVB type-specific p53 mutations (Lehman et al, 1993) as well as some chromosomal abnormalities typical for human skin SCCs (Lehman et al, 1993;Boukamp et al, 1997;Popp et al, 2002). While the HaCaT cells remained nontumorigenic through long-term passaging, they became tumorigenic by transduction with the Harvey-ras oncogene, increased temperature or stroma modulating growth factors known to be relevant for tumorigenicity (Boukamp et al, 1990b(Boukamp et al, , 1995(Boukamp et al, , 1997Skobe and Fusenig, 1998;Obermueller et al, 2004). Thus, the intrinsic stable nontumorigenic phenotype, together with the sensitivity for tumorigenic conversion upon distinct changes, make HaCaT cells a highly suitable model to study the role of UVA in skin carcinogenesis.…”

Mutant p53 subverts p63 control over KLF4 expression in keratinocytes

“…The HaCaT cells analyzed here are immortalized and non-transformed KCs with mutant p53 allelles-R282Q and H179Y-and the levels of the p53 proteins are extremely high. They are not a model for skin cancer, as they are able to differentiate in vitro, unable to form colony assays in soft agar and do not generate tumors in nude mice in vivo (Boukamp et al, 1997;EM and RM, unpublished). However, the p53 missense mutations harbored by HaCaT alleles are indeed UVB signature mutations (Pfeifer and Besaratinia, 2009, and references therein).…”

Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells

“…We have chosen the telomerase-immortalized retinal pigment epithelial cells (RPE hTert) because of their relatively normal and diploid karyotype, compared to cancer cells and other viral-protein immortalized cell types (Boukamp et al, 1997;Duensing and Munger, 2002). RPE hTert only harbors one balanced translocation between chromosomes 2 and X (Figure 5b, i).…”