Generation of a human melanocyte cell line by introduction of HPV16 E6 and E7 genes

Abstract

Availability of a standard human melanocyte cell line with unlimited growth potential and otherwise normal melanocytic properties will greatly facilitate research in melanocyte biology and in vitro studies on the etiology of pigmentary disorders and melanoma. Using a retroviral vector, E6 and E7 open reading frames of human papilloma virus type 16 (HPV 16) have been introduced into cultured normal human melanocytes. Cells selected by increased resistance to geneticin conveyed by the vector and expressing E6E7 mRNA have been cloned to ensure genetic homogeneity. Since their establishment as primary cells, cloned PIG1 cells have undergone more than twice the amount of population doublings of senescent parental cells. Moreover, in passage numbers when parental cells had become senescent, proliferation of clonal cells was retained at levels exceeding those of normal human melanocytes in third passage by 100%. Further characterization has revealed that the cells remain dependent on tetradecanoyl phorbol 13-acetate (TPA) for growth and do not proliferate in soft agar nor form tumors in nude mice. The antigenic profile of the cells was slightly altered as compared to parental cells, but was incomparable to that of M14 melanoma cells. Importantly, PIG1 cells contain more melanin pigment than parental cells.

Exponentially increasing incidences of cutaneous malignant melanoma in Europe correlate with low personal annual UV doses and suggests 2 major risk factors

“…[70][71][72] HPV incorporates into the host's genomic DNA during the DNA damage repair processes [73][74][75] and once incorporated deploys its carcinogenic regime of E6 and E7 proteins that inactivate p53 76 and Bak, 77 respectively, so that the cell cannot die via either the DNA-damage or receptor-initiated apoptotic pathways. 78 However, vitamin D 3 /calcitriol can initiate a p53-independent apoptotic cell death pathway 79 circumventing HPV's plan for cellular immortalization 80 by destroying the infected cell. Note that one of HPV's survival strategies is the production of E2 protein that causes the infected cell to release IL-10 81 helping to conceal HPV from immune surveillance.…”

“…83 In addition, it now appears that HPV can establish a latent infection 84 that may also have consequence both in the UV interaction and in the disruption of normal skin, which has been implicated in melanoma. 85 HPV may represent the first step in the transformation process because it's E6 and E7 proteins immortalize the melanocyte 80 setting it up for accumulating DNA mutations over time and possibly activating HERV, as other viruses are known to do.…”

All sites but skin cancer incidences analyzed worldwide by sex, age, and skin type over time (1955-2007), advancing age, and UVB dose reveals important carcinogenic drivers

“…[15] UVA radiation creates ROS that can further contribute toward carcinogenesis by activating HPV [16] and combined with estrogen cause increased expression of HPV's oncogenic proteins E6 and E7 [17] that immortalize cells by inactivating p53 and pRB, respectively. [18] ROS synergistically increases the risk for getting cancer when combined with estrogen, as shown in a hamster kidney cancer model. [19] Estrogen has also been declared a human carcinogen by the International Agency for Research on Cancer (IARC) [20] because it increases the risk for females getting cervical, breast, ovarian, and endometrial cancers; [21] estrogen also increases the risk for males getting breast [22] and prostate cancers.…”

CRH inhibits NF-κB signaling in human melanocytes

“…The immortalized normal human epidermal melanocyte line PIG1, a gift of Dr. C. Le Poole, Loyola Medical Center, Maywood, IL [19], was maintained at 37 °C in medium 154 with Human Melanocyte Growth Supplement and antibiotics. For experiments PIG1 melanocytes were seeded at 10 000/cm 2 cells in growth medium.…”