p53 Mutations in human immortalized epithelial cell lines

Abstract

Although rodent cells have been immortalized following transfection with a mutant p53 gene, the role of p53 in the immortalization of human cells is unknown. Therefore, human epithelial cell lines were examined for p53 mutations in exons 4-9 which include the evolutionarily conserved regions. A spontaneously immortalized skin keratinocyte cell line, HaCat, and three ras-transfected clones, have a p53 mutational spectrum that is typical of ultraviolet light induced mutations. A normal finite lifespan cell strain (184) and two benzo[a]pyrene immortalized mammary epithelial cell lines derived from 184 (184A1 and 184B5) contain wild type p53 sequences in exons 4-9, although elevated levels of nuclear p53 indicate an alteration in the stability of the normally transient protein. Wild type p53 was found in human bronchial, esophageal and hepatic epithelial cells immortalized by SV40 T antigen gene and human renal epithelial cells immortalized by adenovirus 5. BEAS-2B, an SV40 T antigen immortalized bronchial epithelial cell line and two subclones, have a germline polymorphism at codon 47. Inactivation of p53 by mechanisms such as mutation or complexing with proteins of DNA tumor viruses appears to be important in the immortalization of human epithelial cells.

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

“…UVA irradiation induces DNA damage also in normal human keratinocytes Since the immortal HaCaT keratinocytes harbor p53 mutations (Lehman et al, 1993) which could influence the UVA-dependent damage response, we next investigated normal human skin keratinocytes. G 1 -enriched keratinocytes (Supplementary Figures 3C and D) were irradiated with 60 J/cm 2 UVA and analysed for g-H2AX foci formation directly after irradiation (0 h) as well as after 10, 20, 30, 60 and 360 min (Figure 5a).…”

“…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).…”

Infectivity and expression of the early adenovirus proteins are important regulators of wild-type and ΔE1B adenovirus replication in human cells

“…In these two cases, already after infection with 10 PFU's per cell all cells stained positive for b-galactosidase. By contrast to these two highly infective cell lines the keratinocyte cell line HACAT, also expressing mutant p53 (Lehman et al, 1993), showed a very low infectivity. Even after infection with a MOI of 1000 PFU's per cell only a part of the cells stained positive.…”

Human mammary epithelial cells exhibit a differential p53-mediated response following exposure to ionizing radiation or UV light

“…However, Lehman et al, (1993) had previously sequenced the highly conserved region of p53 known as the mutational hot-spot region (exons 4 ± 9) in HMEC184, HMEC184A1 and HMEC184B5 and found a wildtype p53 sequence. However, p53 mutations have been found outside of the hot-spot region in some breast cancers (Hartmann et al, 1995).…”