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. 2008 Jan 1;68(1):172-80.
doi: 10.1158/0008-5472.CAN-07-2678.

Hypoxia Regulates Choline Kinase Expression Through Hypoxia-Inducible factor-1 Alpha Signaling in a Human Prostate Cancer Model

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Free PMC article

Hypoxia Regulates Choline Kinase Expression Through Hypoxia-Inducible factor-1 Alpha Signaling in a Human Prostate Cancer Model

Kristine Glunde et al. Cancer Res. .
Free PMC article

Abstract

The intensity of the total choline (tCho) signal in spectroscopic images of tumors is spatially heterogeneous. The likewise heterogeneous physiologic tumor microenvironment may contribute to this heterogeneity. We therefore investigated the relationship between hypoxia, choline metabolites, and choline kinase (Chk) in a human prostate cancer model. Human PC-3 prostate cancer cells were engineered to express enhanced green fluorescent protein (EGFP) under hypoxic conditions. These PC-3-5HRE-EGFP cells were characterized in culture and as tumors transplanted in mice using (1)H magnetic resonance spectroscopy (MRS) and MRS imaging (MRSI) combined with EGFP fluorescence microscopy and imaging. Hypoxic EGFP-fluorescing tumor regions colocalized with regions of high tCho in combined MRSI and optical imaging studies. Cellular phosphocholine (PC) and tCho concentrations as well as Chk expression levels significantly increased following exposure of PC-3 cells to hypoxia. A putative promoter region located 5' of the translation start site of the human chk-alpha gene was cloned and luciferase (Luc)-based reporter vector constructs were generated. Luc reporter assays provided evidence that some of the putative hypoxia response elements (HRE) within this putative chk-alpha promoter region functioned in vitro. Chromatin immunoprecipitation assays using an antibody against hypoxia-inducible factor (HIF)-1 alpha showed that HIF-1 can directly bind this region of the endogenous chk-alpha promoter in hypoxic PC-3-5HRE-EGFP cells. These data suggest that HIF-1 activation of HREs within the putative chk-alpha promoter region can increase Chk-alpha expression within hypoxic environments, consequently increasing cellular PC and tCho levels within these environments.

Figures

Figure 1
Figure 1
A, simplified diagram depicting the 5HRE-EGFP construct, which is activated by HIF-1 (HIF-1α/HIF-1β dimer) only under hypoxic conditions as HIF-1α is continuously degraded under normoxic conditions. HIF-1 binds HREs in the 5HRE-EGFP construct resulting in EGFP expression, which can be detected by fluorescence microscopy and imaging. B, bright-field (top) and EGFP fluorescence (bottom) microscopic images (40 × lens) of the same FOVs from normoxic (left) and hypoxic (right) live PC-3-5HRE-EGFP cells where hypoxia induces robust EGFP fluorescence. C, immunoblots of EGFP levels in total protein from cell lysates indicate little or no EGFP expression under normoxia (left lane), whereas high EGFP protein expression was found under hypoxic (right lane) conditions. Actin immunoblotting was performed as a loading control.
Figure 2
Figure 2
A, example of CSI MRSI data set from a PC-3-5HRE-EGFP tumor (550 mm3) obtained with a spatial resolution of 0.5 mm ×0.5 mm ×4.0 mm. B, spectrum from a single 0.5 mm at ×0.5 mm ×4.0 mm voxel showing tCho 3.2 ppm and lactate/triglycerides (lac/triglyc) at 1.3 ppm (TE = 272 ms). C, triplanar view of a tCho map (displayed in red) generated from the data set shown in Fig. 2A, fused with the corresponding spin-echo image displayed in blue. D, EGFP expression in hypoxic regions in a fresh tissue slice matching the MRSI slice, overlaid on a white light image. Comparison of C and D reveals a coarse colocalization between the tCho and EGFP distributions.
Figure 3
Figure 3
A, the warped bright-field image (top left) was used to guide the concurrent warping of the corresponding EGFP fluorescence image (top center). Thus, the hypoxia-induced EGFP fluorescence and the corresponding tCho MRSI data set (top right) were coregistered as described in Materials and Methods. In the resulting overlay image (bottom left), the tCho MRSI image has been rendered in red, whereas EGFP fluorescence is shown in green so that colocalization becomes yellow. Bottom center, colocalization of tCho (red) and EGFP (green) was quantified by generating pixel intensity correlation diagrams of both images. Here, pixels within the yellow rectangular region and bounded by the red lines correspond to the pixels displayed in white in the overlay image shown in the bottom right panel. Therefore, an EGFP fluorescence and tCho MRSI point were only considered as colocalized if their respective intensities were higher than the threshold intensity [e.g., those points with intensities that ranged from 50 to 255 (yellow rectangular) plus an additional intensity ratio threshold of 50% that is bounded by the two red lines]. B, the sum of all pixel intensity correlation diagrams was generated to quantify the colocalization of the EGFP fluorescence (green) with the tCho MRSI signal and vice versa in all 18 tumors. The high number of pixels localized on what would be a straight line defined by y = x indicates good colocalization of EGFP fluorescence and tCho signal. This is indicative of increased tCho levels in tumor regions with high EGFP expression.
Figure 4
Figure 4
Expanded regions of the 1H MR spectra of normoxic (left) and hypoxic (right) PC-3-5HRE-EGFP cells (A) and wild-type PC-3 cells (B) with corresponding immunoblots probed with Chk antibody. Actin immunoblotting was performed as a loading control. C, quantification of 1H MR spectra for wild-type PC-3 (left; n = 3) and PC-3-5HRE-EGFP (right; n = 4) PC-3 cell extracts from cells exposed to 24 h of hypoxic conditions and normoxic controls. Columns, mean; bars, SD. *, P < 0.05; **, P < 0.01.
Figure 5
Figure 5
Schematic diagram of the putative human chk-α promoter region-firefly Luc-based reporter assay constructs. Top, the 2.3-kb region of the human chk-α gene, which is immediately upstream of the translation start site (+1). Putative HRE binding sites (core sequence RCGTG) are labeled HRE 1 to HRE 6 and represented by vertical orange bars. Bottom, P1, entire 2.3-kb Luc reporter construct; P2 to P7, Luc-reported constructs of truncated regions of this 2.3-kb sequence. All constructs were contained in the pGL4-basic vector and tested for endogenous HIF-1–mediated hypoxia-induced Luc expression in transient transfections of wild-type PC-3 and PC-3-5HRE-EGFP cells.
Figure 6
Figure 6
A, the effect of hypoxia on Luc expression from the putative chk-α promoter region constructs, depicted in Fig. 5, was first tested by transient transfection of wild-type PC-3 cells with these constructs followed by exposure of these cells to the hypoxia mimetic CoCl2 (200 μmol/L) for 24 h. Y axis, induction was evaluated as the increase in Luc expression recorded during hypoxia relative to that observed during normoxia. Thus, in case of constructs P1 to P3, Luc expression was repressed under these conditions. Columns, mean (n = 3); bars, SD. *, P < 0.02, hypoxia compared with normoxia. B, wild-type PC-3 cells transiently transfected with the most responsive pGL4-P5-Luc reporter construct were exposed to hypoxic conditions in a hypoxic culture chamber (pO2 < 1%), which resulted in increased Luc activity at 24 h, indicative of HIF-1α stabilization and the apparent binding of HIF-1 to P5. Values are again reported as hypoxia to normoxia ratios. Columns, mean (n = 3); bars, SD. *, P < 0.05, groups were compared as indicated in the figure. C, hypoxic exposure of wild-type PC-3 (black columns) and PC-3-5HRE-EGFP (gray columns) cells transfected with pGL4-P5-Luc resulted in an induction of Luc activity at 24 h. Columns, mean (n = 2–3); bars, SD. *, P < 0.05, groups were compared as indicated in the figure. D, direct hypoxia-induced binding of HIF-1 to a region within the putative human chk-α promoter was shown in PC-3 cells by ChIP. Following normoxia or hypoxia (hypoxic culture chamber; pO2 < 1%), cross-linked chromatin-protein complexes from the treated PC-3 cells were immunoprecipitated with anti-HIF-1α antibody or control antibodies. A 282-bp sequence within the putative chk-α promoter, encompassing HREs 3 to 5, as depicted in Fig. 5, was PCR amplified from these precipitates using the primers ChIP S-1 and ChIP AS-1 (see Materials and Methods). Lane 1, total unprocessed chromatin; lane 2, anti-HIF-1α antibody precipitation following hypoxia; lane 3, anti-histone deacetylase 3 antibody precipitation following hypoxia; lane 4, anti-GAPDH antibody precipitation following hypoxia; lane 5, anti-HIF-1α precipitation following normoxia. Identical volumes from the final precipitations were used in the PCR.

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