Lack of functional retinoblastoma protein mediates increased resistance to antimetabolites in human sarcoma cell lines.

Abstract

Growth inhibition assays indicated that the IC50 values for methotrexate (MTX) and 5-fluorodeoxyuridine (FdUrd) in HS-18, a liposarcoma cell line lacking retinoblastoma protein (pRB), and SaOS-2, an osteosarcoma cell line with a truncated and nonfunctional pRB, were 10-to 12-fold and 4-to 11-fold higher, respectively, than for the HT-1080 (fibrosarcoma) cell line, which has wild-type pRB. These Rb-/-cell lines exhibited a 2-to 4-fold increase in both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) enzyme activities as well as a 3-to 4-fold increase in mRNA levels for these enzymes compared to the HT-1080 (Rb+/+) cells. This increase in expression was not due to amplification of the DHFR and TS genes. Growth inhibition by MTX and FdUrd was increased and DHFR and TS activities and expression were correspondingly decreased in Rb transfectants of SaOS-2 cells. In contrast, there was no significant difference in growth inhibition among these cell lines for the nonantimetabolites VP-16, cisplatin, and doxorubicin. A gel mobility-shift assay showed that parental SaOS-2 cells had increased levels of free E2F compared to the Rb-reconstituted SaOS-2 cells. These results indicate that pRB defective cells may have decreased sensitivity to growth inhibition by target enzymes encoded by genes whose transcription is enhanced by E2F proteins and suggest mechanisms of interaction between cytotoxic agents and genes involved in cell cycle progression.polyglutamate formation or impaired uptake (17). The fifth cell line (HS-18), although able to transport MTX and make long-chain polyglutamates comparable to the MTX-sensitive HT-1080 fibrosarcoma cell line, had an increased level of the MTX target enzyme DHFR compared to the other cell lines, without evidence of DHFR gene amplification. The subsequent finding that this cell line lacked both copies of chromosome 13, which contains the gene for pRB (18), led us to investigate further the relationship between pRB and resistance to MTX as well as FdUrd, antimetabolite drugs that inhibit enzymes involved in DNA replication.This study addresses the relationship between absent or abnormal pRB and sensitivity to drugs that target DHFR and TS. We have compared the growth inhibition of a human sarcoma cell line containing pRB (HT-1080) with cell lines either lacking pRB (HS-18) or having a truncated pRB incapable of binding E2F (SaOS-2). We have also obtained viable SaOS-2 sublines stably transfected with Rb after G-418 selection (see below). Cells lacking functional pRB were more resistant to growth inhibition by MTX and FdUrd compared to pRB-expressing cell lines. In cell lines lacking pRB, levels of DHFR and TS enzyme activity as well as mRNA expression were higher. Furthermore, cell lines lacking pRB had increases in free E2F as measured by gel-retardation assays.A wide variety of human cancers demonstrate loss or mutation in the tumor suppressor gene p53 and the retinoblastoma tumor suppressor gene (Rb) (reviewed in refs. 1 and 2). Rb encodes a nuclear phosphoprotein...

Molecular Mechanisms Underlying the Synergistic Interaction of Erlotinib, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, with the Multitargeted Antifolate Pemetrexed in Non-Small-Cell Lung Cancer Cells

“…However, the basal expression of EGFR and TS was also assessed in this study to evaluate the effect of 72-h treatment with IC 50 levels of erlotinib, pemetrexed, and their combination in all cell lines. Because previous studies have shown a strong correlation between expression levels of TS and its upstream transcriptional regulator E2F-1 (Li et al, 1995;Huang et al, 2007), and because other studies showed that EGFR-TKIs affected E2F-1 (Kobayashi et al, 2006;Suenaga et al, 2006), we also evaluated the expression of E2F-1 mRNA. Moreover we studied the expression levels of DHFR, GARFT, RFC, ␥-glutamyl hydrolase (␥GH), and folyl-polyglutamate synthetase (FPGS).…”

RB family members as predictive and prognostic factors in human cancer

“…Lack of functional Rb protein appears to mediate increased resistance to antimetabolites in human sarcoma cell lines (Li et al, 1995).…”

Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents

“…This is attributed to elevated levels of the metabolic enzymes targeted by these compounds (TS, dihydrofolate reductase and ribonucleotide reductase (RNR)) Li et al, 1995;Angus et al, 2002). Therefore, we next probed the consequences of acute RB loss on the response of primary and immortal cultures to antimetabolites.…”

“…However, TS inhibition is believed to be the principle mechanism of action of 5-FU (Peters et al, 2002). TS is a well established target of the RB/E2F signaling axis DeGregori et al, 1995;Li et al, 1995), and RB-deficient cell lines display increased TS expression and activity that is associated with resistance to agents that target TS . Therefore, we next sought to determine the effects of acute RB loss on TS expression in primary MAFs and how this contributes to the observed lack of cell cycle checkpoint response to 5-FU in RB-deficient MAFs.…”