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Hypoxia and apoptosis in colon cancer
Başlık:
Hypoxia and apoptosis in colon cancer
Yazar:
Erler, Janine, author.
ISBN:
9780438085121
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (216 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Özet:
In solid tumours, such as colon cancer tumours, cells are subjected to hypoxic conditions due to insufficient blood supply. Hypoxic tumour cells are resistant to radiotherapy and some forms of chemotherapy. This thesis examines the effect of low oxygen on the Bcl-2 family that play a central role in determining the cellular threshold for the induction of apoptosis, and assesses the impact of this on cellular response to therapy. Cells from a panel of human colon carcinoma cell lines (HCT116, HT29 and SW480) were incubated under conditions of normoxia, hypoxia (1% oxygen) or anoxia (<0.1% oxygen) for up to 18 hours. Cellular response to chemotherapy, cell death, cell cycle profile, clonogenicity and changes in Bcl-2 family mRNA and protein expression levels were assessed. Short-term viability tests and clonogenic assays showed that oxygen deprivation resulted in resistance to a variety of clinically relevant chemotherapeutic drugs, although incubation of cells from the panel of cell lines under anoxic conditions for 16h itself had no effect on cell viability, clonogenicity, cell cycle distribution or S-phase traverse. However, anoxic incubation resulted in decreased levels of pro-apoptotic Bcl-2 family proteins Bid, Bax, Bad, Bim and Bnip3 occurring within 8 hours of exposure. In contrast, the levels of the anti-apoptotic proteins Bcl-2, Bcl-xl and Bcl-w remained unchanged. Quantitative RT-PCR analyses indicated that the observed decreases in pro-apoptotic protein expression occurred at the mRNA level albeit to different degrees. Incubation of each cell line under anoxic conditions resulted in increased levels of HIF-1alpha protein and of the HIF-1 transcriptional targets VEGF and CA-IX proteins suggesting the colon lines express functional HIF-1. This was confirmed using a dual luciferase assay designed to test for HIF-1 functionality. Anoxia-induced down-regulation of pro-apoptotic proteins was also seen in HEPA-1, CHO, V79 and MEF cells indicative of a general effect of anoxia rather than a specific effect in colon carcinomas. Comparison of HEPA-1 and CHO HIF-1 proficient and deficient cell line pairs demonstrated that anoxia-induced down-regulation of Bid is HIF-1 dependent. This was consistent with the absence of anoxia-induced Bid down-regulation in HT29 cells in which HIF-1alpha expression was ablated with RNAi, and with the discovery of sequences consistent with hypoxia response elements in the Bid promoter sequence. DNA damage of normoxic HCT116 cells (containing wild type p53) provoked p53 accumulation and upregulation of Bax and p21, yet when these cells experienced anoxia Bax and p21 levels decreased despite p53 accumulation. The contribution of Bid and/or Bax down regulation to drug responsiveness was demonstrated by the relative resistance of normoxic cells that had no or reduced expression of Bid and/or Bax, and by significantly increased sensitivity to etoposide in hypoxic cells with maintained high levels of Bid expression. These data couple hypoxia and cell survival with the p53-independent down- regulation of key pro-apoptotic Bcl-2 family proteins, and for Bid this occurs via a HIF-1 dependent transcriptional mechanism, adding impetus to the design of chemotherapeutic strategies to target this transcription factor. The data provide a mechanistic explanation for pleiotropic drug resistance in hypoxic tumour cells.
Notlar:
School code: 1543
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(686930.1) | 686930-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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