Impact of VKORC1, CYP2C9, and CYP4F2 Polymorphisms on Optimal Warfarin Dose: Does Ethnicity Matters?

BACKGROUND: Conventional anticoagulation with warfarin remains the cornerstone strategy for numerous preventive strategies. It is established that Asian patients require lower warfarin doses than Caucasians potentially attributing to the genetic polymorphism (GP) differences. AREAS OF UNCERTAINTY: The impact of GP on optimal warfarin dose (OWD) in Koreans is unclear when compared with other ethnicities. It is also not well established whether GP linked to OWD in Korean patients to the similar extend as in Chinese, Japanese, and Caucasians. DATA SOURCES: Single-center prospective observational study in Koreans, matched with historic cohorts of other ethnicities. THERAPEUTIC ADVANCES: Clinical characteristics, concomitant medications, OWD, international normalized ratio, and VKORC1, CYP2C9, and CYP4F2 GPs were assessed in consecutive Korean patients. The OWD was defined when patient's international normalized ratio was within target range for at least 3 consecutive times separated by 1 week. We included 133 (mean age 62.6 ± 12.1 years, 49% males) warfarin-treated patients of Korean descend. The mean OWD was 3.30 ± 1.34 (range: 1-9) mg/d. Homozygous wild-type patients required lower OWD (3.1 ± 1.1 mg/d vs. 4.7 ± 1.8 mg/d, P < 0.001) for VKORC1 and higher OWD for both CYP2C9 (3.4 ± 1.3 mg/d vs. 2.3 ± 1.1 mg/d, P = 0.002) and CYP4F2 (3.0 ± 1.2 mg/d vs. 3.4 ± 1.3 mg/d vs. 4.0 ± 1.7 mg/d, P = 0.033) than those carrying heterozygote genes. CONCLUSIONS: Korean patients exhibit different VKORC1, CYP2C9, and CYP4F2 profiles impacting lower OWD in Eastern Asians than required in Caucasians. Universal international OWD guidelines may consider patient ethnicity as a confounder; however, this hypothesis needs further clarification.

K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling.

Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the mechanism by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this mechanism. We have used a model of telomerase-immortalized human pancreatic duct-derived cells (E6/E7/st) to study mechanisms of Ras growth transformation. First, we found that human papillomavirus E6 and E7 oncogenes, which block the function of the p53 and Rb tumor suppressors, respectively, and SV40 small t antigen were required to allow mutant K-Ras(12D) growth transformation. Second, K-Ras(12D) caused growth transformation in vitro, including enhanced growth rate and loss of density dependency for growth, anchorage independence, and invasion through reconstituted basement membrane proteins, and tumorigenic transformation in vivo. Third, we determined that the Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide exchange factor effector pathways were activated, although extracellular signal-regulated kinase (ERK) activity was not up-regulated persistently. Finally, pharmacologic inhibition of Raf/mitogen-activated protein kinase/ERK and PI3K signaling impaired K-Ras-induced anchorage-independent growth and invasion. In summary, our studies established, characterized, and validated E6/E7/st cells for the study of Ras-induced oncogenesis.

Ras-driven transformation of human nestin-positive pancreatic epithelial cells.

Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the means by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this signaling biology. This chapter describes the establishment and characterization of telomerase-immortalized human pancreatic duct-derived cells to study mechanisms of Ras growth transformation. An important strength of this model system is the ability of mutationally activated K-Ras to cause potent growth transformation in vitro and in vivo. We have utilized this cell system to evaluate the antitumor activity of small molecule inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase cascade. This model will be useful for genetic and pharmacologic dissection of the contribution of downstream effector signaling in Ras-dependent growth transformation.

Notch 2-positive progenitors with the intrinsic ability to give rise to pancreatic ductal cells.

Pancreatic adenocarcinomas display foci of duct-like structures that are positive for markers of pancreatic ductal cells. The development of these tumors is promoted by conditions leading to acinar-to-ductal metaplasia, a process by which acinar cells are replaced by ductal cells. Acinar-to-ductal metaplasia has recently been shown to proceed through intermediary cells expressing Nestin. To create an in vitro system to study pancreatic adenocarcinomas, we had used an hTERT cDNA to immortalize primary cells of the human pancreas. In this report, we show that the immortalized cells, termed hTERT-HPNE cells, have the ability to differentiate to pancreatic ductal cells. Exposing hTERT-HPNE cells to sodium butyrate and 5-aza-2'-deoxycytidine lead to the formation of pancreatic ductal cells marked by the expression of MDR-1, carbonic anhydrase II, and the cytokeratins 7, 8, and 19. hTERT-HPNE cells were found to have properties of the intermediary cells formed during acinar-to-ductal metaplasia, which included their undifferentiated phenotype, expression of Nestin, evidence of active Notch signaling, and ability to differentiate to pancreatic ductal cells. These results provide further evidence for the presence in the adult pancreas of a precursor of ductal cells. hTERT-HPNE cells should provide a useful model to study acinar-to-ductal metaplasia and the role played by this process in pancreatic cancer development.

Use of exogenous hTERT to immortalize primary human cells.

A major obstacle to the immortalization of primary human cells and the establishment of human cell lines is telomere-controlled senescence. Telomere-controlled senescence is caused by the shortening of telomeres that occurs each time somatic human cells divide. The enzyme telomerase can prevent the erosion of telomeres and block the onset of telomere-controlled senescence, but its expression is restricted to the early stages of embryonic development, and in the adult, to rare cells of the blood, skin and digestive track. However, we and others have shown that the transfer of an exogenous hTERT cDNA, encoding the catalytic subunit of human telomerase, can be used to prevent telomere shortening, overcome telomere-controlled senescence, and immortalize primary human cells. Most importantly, hTERT alone can immortalize cells without causing cancer-associated changes or altering phenotypic properties. Primary human cells that have so far been established by the forced expression of hTERT alone include fibroblasts, retinal pigmented epithelial cells, endothelial cells, oesophageal squamous cells, mammary epithelial cells, keratinocytes, osteoblasts, and Nestin-positive cells of the pancreas. In this article, we discuss the use of hTERT to immortalize of human cells, the properties of hTERT-immortalized cells, and their applications to cancer research and tissue engineering.