Puuttuuko p53-funktio pankreaasta? Mikä sen immuunisysteemi on?

Am J Pathol. 2000 Nov; 157(5): 1623–1631.

doi:  10.1016/S0002-9440(10)64800-6

PMCID: PMC1885733

Immortal Human Pancreatic Duct Epithelial Cell Lines with Near Normal Genotype and Phenotype

Hong Ouyang,^* Lun-jun Mou,^* Catherine Luk,^† Ni Liu,^* Jana Karaskova,^* Jeremy Squire,^*†‡ and Ming-Sound Tsao^*†‡

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Abstract

Immortal epithelial cell lines were previously established after transduction of the HPV16-E6E7 genes into primary cultures of normal pancreatic duct epithelial cells. Single clones were isolated that demonstrated near normal genotype and phenotype. The proliferation of HPDE6-E6E7c7 and c11 cells is anchorage-dependent, and they were nontumorigenic in SCID mice. The cell lines demonstrated many phenotypes of normal pancreatic duct epithelium, including mRNA expression of carbonic anhydrase II, MUC-1, and cytokeratins 7, 8, 18, and 19. These cells have normal Ki-ras, p53, c-myc, and p16^INK4A genotypes. Cytogenetic studies demonstrated losses of 3p, 10p12, and 13q14, the latter included the Rb1 gene. The wild-type p53 protein was detectable at very low levels consistent with the presence of E6 gene product, and the lack of functional p53 pathway was confirmed by the inability for γ-irradiation to up-regulate p53 and p21waf1/cip1 protein. The p110/Rb protein level was also not detectable consistent with the expression of E7 protein and haploid loss of Rb1 gene. Despite this, the proliferation of both c7 and c11 cells were markedly inhibited by transforming growth factor-β1. This was associated with up-regulation of p21cip1/waf1 but not p27kip1. Further studies showed that p130/Rb2 and cyclin D3 were expressed, suggesting that p130/Rb2 may have partially assumed the maintenance of G₁ cell cycle checkpoint regulation. These results indicate that except for the loss of p53 functional pathway, the two clones of HPDE6-E6E7 cells demonstrated a near normal genotype and phenotype of pancreatic duct epithelial cells. These cell lines will be useful for future studies on the molecular basis of pancreatic duct cell carcinogenesis and islet cell differentiation.

Pancreatic cancer is the fourth commonest cause of cancer death in North America and has one of the worst prognoses. ¹ Greater than 90% of these tumors arise from the pancreatic duct epithelium. These tumors are highly metastatic and only 20% of the patients are treated by surgical resection. ² Even when the primary tumor is small and localized, the prognosis remains poor and chemotherapy or radiotherapy has demonstrated limited effectiveness. ^3,4 Therefore, it seems that a significant improvement in pancreatic cancer mortality depends on the development of better treatment and preventive strategies, which require knowledge on the molecular biology and pathogenesis of this disease. Recent definitions of genetic changes that occur commonly in pancreatic cancer represent important first steps toward such a goal. ⁵ Nevertheless, the availability of dynamic models remains crucial to the study and understanding of the biological significance of these genetic changes, especially in the context of pancreatic duct epithelial cell carcinogenesis.

Our laboratory has previously reported the establishment of primary and immortal epithelial cell lines from normal human pancreatic ducts. ⁶ We also reported that in comparison with the pancreatic cancer cell lines, the human pancreatic duct epithelial (HPDE) cells demonstrated a gene expression pattern that more consistently resembled the phenotype of normal cells rather than cancerous duct cells in vivo. ⁷ These similarities included relatively low expression levels of various tyrosine kinase receptors, a wild-type Ki-ras genotype, and the retention and expression of p16INK4A gene. We have subsequently isolated several clones of these cell lines. We report here the phenotypic and genotypic characteristics of two of these cell lines that demonstrate anchorage-dependent growth requirement and that are nontumorigenic in immune-deficient mice.