PMC

a) KLN-tTA/pBISA-DL^(ATF-4) cells were transfected with mock siRNA or siRNA targeting PKR, PERK, GCN2, or HRI individually or simultaneously in all combinations (only PKR, PERK, and GCN2 combination is shown). CRL-2813 cells were transfected in the same manner except that the transfection mixture also contained the pBISA-DL^(ATF-4) and tTA plasmids. Cells were treated with compound BTdCPU or with DMSO and the normalized F/R ratio was determined by DLR. b) KLN-tTA/pBISA-DL^(ATF-4) or CRL-2813 cells were transfected with siRNAs targeting each of the eIF2α kinases and treated with compound BTdCPU or with DMSO. Expression of CHOP mRNA was determined by real-time PCR. c) CRL-2813 cells were transfected with mock, PERK or HRI siRNA, treated with tunicamycin, compound BTdCPU or vehicle, and the levels of phosphorylated (p-eIF2α) and total eIF2α (eIF2α) were determined by Western blot. d) KLN-tTA/pBISA-DL^(ATF-4) cells were transfected with mock or HRI-targeting siRNA, treated with four N,N’-diarylurea compounds or vehicle and the normalized F/R ratio was determined by DLR. e) CRL-2813 cells were transfected with mock or HRI targeting siRNA, treated with four N,N’-diarylurea compounds or vehicle and the normalized F/R ratio was determined by DLR.

a) The PC-3 human prostate cancer cells in which endogenous eIF2α is replaced by recombinant WT or the non-phosphorylatable eIF2α-S51A mutant were treated with the indicated concentrations of N,N’-diarylureas and cell proliferation was measured by SRB assay. Panel a shows the growth inhibition curve for one active (BTCtFPU) and one inactive (NCPdCPU) N,N’-diarylurea. Calculated IC₅₀ for all four compounds in these genetically engineered cell lines are shown in ) CRL-2813 human melanoma cancer cells were transfected with HRI or mock siRNA, treated with the indicated concentrations of N,N’-diarylureas and cell proliferation was measured by SRB assay. The panel b shows the growth inhibition curve for one active (BTCtFPU) and one inactive (NCPdCPU) N,N’-diarylurea, calculated IC₅₀ for all four compounds in cells transfected with HRI or mock siRNA is shown in . The experiment was conducted in triplicates and each experiment was independently performed three times.

a) KLN-tTA/pBISA-DL^(ATF-4) (left) or PC-3 cell (right) lines were incubated with selected N,N’-diarylureas, levels of phosphorylated (p-eIF2α) and total eIF2α (eIF2α) were determined by Western blot analysis with pS51-eIF2α specific rabbit monoclonal antibodies or with total eIF2α specific mouse monoclonal antibodies; respectively. b) The PC-3 cells in which endogenous eIF2α is replaced by recombinant WT or non-phosphorylatable eIF2α-S51A mutant were co-transfected with tTA and pBISA-DL^(ATF-4) dual luciferase expression vector and treated with the indicated concentrations of N,N’-diarylureas. The normalized F/R ratio was determined by DLR assay and standard error of mean are shown. c) Genetically engineered PC-3 cells in (b) were treated with N,N’-diarylureas (10 μM) and the expression of CHOP mRNA was determined by real-time PCR. The experiment was conducted in triplicates and each experiment was independently performed three times.

a) F-luc and R-luc ORFs were cloned into pBISA plasmid to transcribe two reporter mRNAs. The 5’UTR of the mouse ATF-4 mRNA including first two codons of bona-fide ORF was cloned in frame with respect to the start codon of F-luc ORF (pBISA-DL^(ATF-4)). The mRNA products of pBISA-DL^(ATF-4) plasmid are shown. b) The structure of three active (a) and one inactive (i) N,N’-diarylureas. c) KLN-tTA/pBISA-DL^(ATF-4) cells were incubated with the indicated concentrations of each N,N’-diarylurea and the normalized F/R ratio was determined by DLR assay. d) KLN-tTA/pBISA-DL^(ATF-4) cells were incubated with the indicated concentrations of each N,N’-diarylurea and expression of endogenous CHOP protein was determined by Western blot analysis. e) KLN-tTA/pBISA-DL^(ATF-4) cells were incubated with 5 or 20 μM of each N,N’-diarylurea and expression of endogenous CHOP mRNA was determined by real-time PCR. 3 replicates in each experimental and control group and each experiment was independently performed 3 times.

a) Lysates were prepared from KLN mouse squamous cell carcinoma, HTB-26, HTB-128, and CRL-2351 human breast, PC-3 human prostate, and CRL-2813 human melanoma cancer cell lines were separated by SDS-PAGE and probed with antibodies specific to HRI or β-actin and quantified. The concentration of the three active N,N’-diarylureas that inhibit proliferation of these cells by 50% (IC₅₀) were plotted against the levels of HRI (normalized for β-actin levels) in the cancer cell lines. Each experiment was independently performed three times. b) Five female nude mice each were treated with 200 mg/kg/d, 100 mg/kg/d and 350 mg/kg/d BTdCPU in 15 μl DMSO or 15 μl DMSO daily for seven days, weighed every other day for total of 15 days and then necropsy was performed. The average body weight of each group is plotted against the time. c) Female nude mice xenografted with MCF-7 human breast cancer cells were randomly distributes to two groups and treated with 175 mg/kg/d BTdCPU in 15 μl DMSO or DMSO alone. Mice were observed daily, and tumor dimensions were measured weekly. d) Lysates prepared from three excised tumors in the treatment and control groups, separated by the SDS-PAGE and blotted with antibodies specific to phosphorylated (P-eIF2α) or total eIF2α (eIF2α) and ratio of phosphorylated eIF2α to total eIF2α was quantified (see ).