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. 2012 Feb;11(1):67-76.

Monitoring the induction of heat shock factor 1/heat shock protein 70 expression following 17-allylamino-demethoxygeldanamycin treatment by positron emission tomography and optical reporter gene imaging

Mikhail Doubrovin  1 , Jian T Che, Inna Serganova, Ekaterina Moroz, David B Solit, Lyudmila Ageyeva, Tatiana Kochetkova, Nagavarakishore Pillarsetti, Ronald Finn, Neal Rosen, Ronald G Blasberg
Affiliations

Monitoring the induction of heat shock factor 1/heat shock protein 70 expression following 17-allylamino-demethoxygeldanamycin treatment by positron emission tomography and optical reporter gene imaging

Mikhail Doubrovin et al. Mol Imaging. 2012 Feb.

Abstract

The cell response to proteotoxic cell stresses is mediated primarily through activation of heat shock factor 1 (HSF1). This transcription factor plays a major role in the regulation of the heat shock proteins (HSPs), including HSP70. We demonstrate that an [124I]iodide-pQHNIG70 positron emission tomography (PET) reporter system that includes an inducible HSP70 promoter can be used to image and monitor the activation of the HSF1/HSP70 transcription factor in response to drug treatment (17-allylamino-demethoxygeldanamycin [17-AAG]). We developed a dual imaging reporter (pQHNIG70) for noninvasive imaging of the heat shock response in cell culture and living animals previously and now study HSF1/HSP70 reporter activation in both cell culture and tumor-bearing animals following exposure to 17-AAG. 17-AAG (10-1,000 nM) induced reporter expression; a 23-fold increase was observed by 60 hours. Good correspondence between reporter expression and HSP70 protein levels were observed. MicroPET imaging based on [124I]iodide accumulation in pQHNIG70-transduced RG2 xenografts showed a significant 6.2-fold reporter response to 17-AAG, with a corresponding increase in tumor HSP70 and in tumor human sodium iodide symporter and green fluorescent protein reporter proteins. The HSF1 reporter system can be used to screen anticancer drugs for induction of cytotoxic stress and HSF1 activation both in vitro and in vivo.

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Figures

Figure 1
Figure 1
Growth profile of RG2-pQHNIG70 cells (A). A 17-AAG concentration-dependent inhibition of RG2-pQHNIG70 cell growth is shown. The growth profiles were fitted to the Gompertz equation [31]. Induction kinetics of GFP expression (B and C). RG2-pQHNIG70 cells were exposed to 17-AAG for 24 hours at the indicated concentrations, and GFP intensity was assessed by FACS analysis and fluorescence microscope at the indicated times (B). The percentage of GFP(+) cells in the M3 region of the FACS profiles shown in panel B was determined (C). Values represent the mean± SD (n=3). Induction of hNIS and HSP70 expression (D and E). Immunocytochemical analysis of hNIS expression in RG2-pQHNIG70 cells (D) following no 17-AAG exposure (a) and following 17-AAG exposure for 24 hours at concentrations of 10 nM (b), 100 nM (c), and 1000 nM (d). Immunoblot analysis of inducible HSP70 protein expression in RG2-pQHNIG70 cells treated with 17-AAG (E). HSP70 levels after a 24 and 48 hour exposure to different concentrations of 17-AAG are shown. Induction of iodide and pertechnetate uptake (F). Radiotracer uptake in RG2 wild-type and RG2-pQHNIG70 cells was determined after a 24 hour exposure to 17-AAG at different concentrations. The 10 minute cell/medium uptake ratio (ml medium/g cells) is shown for 99mTcO4-pertechnetate (a) and 131I-iodide (b); values represent the mean ± SD (n=3).
Figure 2
Figure 2
Relationship between iodide and pertechnetate uptake with respect to HSP70 protein and eGFP fluorescence of RG2-pQHNIG70 cells (A and B). The results shown in Panel A compare the data presented in Fig. 1B and Fig 1F; the regression equation for iodide (solid squares) was y = 0.83 + 0.12 x, R2 = 0.844, and for 99mTcO4− (solid circles) it was y = 0.97 + 0.46 x, R2 = 0.840. Panel B compares the data presented in Figure 1E and 1F; the intensity of the HSP70 immunoblot is expressed in arbitrary units. The regression equation for iodide (solid squares) was y = 0.76 + 0.0014x, R2 = 0.939, and for 99mTcO4− (solid circles) it was y = 0.63 + 0.0054x, R2 = 0.949. Relationship between eGFP fluorescence and HSP70 protein in RG2-pQHNIG70 cells (C). The results shown in Fig. 1B and 1 are compared; the regression equation is y = 2.9 + 0.0090x, R2=0.815.
Figure 3
Figure 3
([124I]-iodide microPET imaging of pQHNIS70 xenografts (A). The coronal and axial microPET images of [124I]-iodide uptake in a representative non-treated (a) and 17-AAG-treated (c) animal are shown; a photograph (b) is also shown. The yellow dashed line on the coronal images and photograph shows the approximate plane of the axial slice. Each animal had three s.c. xenografts; a test RG2-pQHNIG70 was located in the left shoulder (red outline), a positive control RG2-pQCNIG xenograft [27] was located in the right shoulder (green outline), and a negative control RG2 wild-type xenograft was located in the right thigh (blue outline). The 17-AAG-treated animals received 150 mg/kg of 17-AAG i.p. 24 hours prior to imaging. A 10 minute microPET image acquisition was obtained 120 minutes after i.v. injection of 100 μCi (3.7 MBq) of 124I−. Western blot for HSP70 protein (B). The RG2-pQHNIG70 xengrafts shown in Panel A were processed for immunoblot with rabbit polyclonal anti-hHSP70 antibody. Immunocytochemical analysis (C). The RG2-pQHNIG70 xenograft tissue obtained from the animal shown in Panel A was fixed and immunostained with antibodies specific for HSP70, hNIS and eGFP.
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