Establishment and characterization of monoclonal 5-fluorouracil-resistant cell lines derived from human endometrial adenocarcinoma

Abstract

Abstract. To investigate acquired 5-fluorouracil (5FU)-resistance in cancer cells, we established four monoclonal 5FU-resistant cell lines from human endometrial adenocarcinoma cells by long-term 5FU-exposure cultures and limiting dilution cultures. The established subclones exhibited 5-25 times greater 5FU-resistance than the parent cells, and showed suppression of 5FU-induced DNA fragmentation. All four 5FU-resistant subclones were 25-125 times more resistant to SN38, 4-hydroxy-cyclophosphamide, paclitaxel and etoposide than the parent cells while none of the four subclones showed resistance to mitomycin. Two of the four subclones showed no resistance to pirarubicin and bleomycin. Although all four 5FU-resistant subclones were 5-25 times more resistant to anti-Fas IgM than the parent cells, the resistance levels to anti-Fas IgM of the individual subclones did not coincide with the strengths of their multidrug resistance. Karyotyping analyses revealed that the parent cells and the three 5FU-resistant subclones examined had normal 46XX karyotypes. These results indicate that the cell death signals induced by mitomycin, pirarubicin and bleomycin are distinctly different from the Fas-mediated apoptotic signals, that acquisition of 5FU-resistance can occur without any large chromosomal deletions or rearrangements, and that there are several possible molecular changes during the acquisition of 5FU-resistance. The established cell lines represent useful tools for investigating the mechanisms and treatments of acquired 5FU-resistance.

Impaired death-associated protein kinase-mediated survival signals in 5-fluorouracil-resistant human endometrial adenocarcinoma cells

“…Since DAPK-KD induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HHUA cells (15), it is highly possible that DAPK-mediated signals constitutively suppress the tumor necrosis factor receptor-mediated cell death signaling common to the Fas-mediated and TRAIL-mediated pathways. Although typical apoptosis with DNA fragmentation can easily be induced in HHUA cells by treatment with 5-fluorouracil (5FU) (19), DAPK-KD increases apoptotic susceptibility to 5FU in HHUA cells (20). However, DAPK-KD has no effect on etoposide (VP16)-sensitivity in HHUA cells (20).…”

“…DAPK constitutively suppresses 5FU-stimulated cell death in the parent HHUA cells, while all the 5FU-resistant subclones established from HHUA cells show not only 5FU-resistance but also crossresistance to various anticancer drugs (19). The 5FU-resistant subclones also show significantly reduced sensitivity to Fas-mediated apoptosis.…”

“…Using three monoclonal 5FU-resistant subclones derived from HHUA cells (5FUr3C, 5FU3D and 5FUr4A) that show multidrugresistance (19), the effects of DAPK-KD on the 5FU-sensitivity of the subclones were analyzed. A previous report demonstrated that transfection of the two DAPK siRNAs (DAPK siRNA1 and siRNA2) used in the study strongly and specifically suppresses DAPK protein expression in HHUA cells (17).…”

“…To clarify the molecular mechanisms of acquired 5FU-resistance in cancer cells, we previously established several monoclonal 5FU-resistant subclones from HHUA cells (19). DAPK constitutively suppresses 5FU-stimulated cell death in the parent HHUA cells, while all the 5FU-resistant subclones established from HHUA cells show not only 5FU-resistance but also crossresistance to various anticancer drugs (19).…”

STAT3 enhances intracellular Fas-mediated apoptotic signals in HHUA human endometrial epithelial cells

“…in the present study, the HHua human endometrial epithelial cell line was selected because the cells express high levels of Fas antigen as well as functional estrogen receptors and progesterone receptors, similar to the normal human endometrial epithelium during the implantation period (17), and form glandular luminal structures in collagen gel cultures, similar to the structures of normal glandular epithelial cells (18). karyotyping analysis performed on 20 HHua cells revealed normal 46XX karyotypes (19). HHua cells are known to express functional Fas antigens on their cell surface that mediate specific apoptotic signals (9).…”