Targeting focal adhesion turnover in invasive breast cancer cells by the purine derivative reversine.

BACKGROUND: The dynamics of focal adhesion (FA) turnover is a key determinant for the regulation of cancer cell migration. Here we investigated FA turnover in a panel of breast cancer models with distinct invasive properties and evaluated the impact of reversine on this turnover in relation to cancer cell invasion in in vitro and in vivo conditions. METHODS: Live imaging and immunofluorescence assays were used to investigate FA turnover in breast cancer cells. Biochemical studies were used to investigate the impact of reversine on FA signalling and turnover. In vivo activity was investigated using orthotopic breast cancer mouse models. RESULTS: Accelerated FA disassembly from plasma membrane protrusions was observed in invasive compared with non-invasive breast cancer cells or non-immortalised mammary epithelial cells. Reversine significantly inhibited FA disassembly leading to stable FAs, which was associated with reduced cell motility and invasion. The inhibitory effect of reversine on FA turnover accounted for a large part on its capacity to interfere with FAK function on regulating its downstream targets. In orthotopic breast cancer mouse models, reversine revealed a potent inhibitory activity on tumour progression to metastasis. CONCLUSION: These results support the utility of targeting FA turnover as a therapeutic approach for invasive breast cancer.

Reduced in vivo lung metastasis of a breast cancer cell line after treatment with Herceptin mAb conjugated to chemotherapeutic drugs.

Anthracyclines and taxanes have remarkable anticancer efficacy, but have poor selectivity and high toxicity. Targeted delivery of chemotherapeutics has emerged as a strategy to achieve higher drug levels at the tumor site, to spare noncancerous tissue and potentially to use lower systemic drug doses, thus preventing side effects. In this study, we targeted the HER2 receptor using the monoclonal antibody (mAb) Herceptin (Trastuzumab) chemically conjugated to Doxorubicin or Taxol. In vitro, drug-Herceptin conjugates exhibited cytotoxicity comparable to equimolar concentrations of free drugs, with the benefit that the cytotoxicity of the conjugates was selective for cells expressing the HER2 target. In vivo, treatment of tumor-bearing mice with Taxol-Herceptin conjugates had a reduction of primary tumors comparable to equivalent doses of free drugs. However, Taxol-Herceptin conjugates significantly reduced metastasis compared with equivalent doses of free drugs. Thus, the data support the concept that conjugates might target metastasis better than primary tumors. This would offer a potential therapeutic approach for management of metastatic breast cancer.

Regulation of focal adhesion turnover by ErbB signalling in invasive breast cancer cells.

A crucial early event by which cancer cells switch from localised to invasive phenotype is initiated by the acquisition of autonomous motile properties; a process driven by dynamic assembly and disassembly of multiple focal adhesion (FA) proteins, which mediate cell-matrix attachments, extracellular matrix degradation, and serve as traction sites for cell motility. We have reported previously that cancer cell invasion induced by overexpression of members of the ErbB tyrosine kinase receptors, including ErbB2, is dependent on FA signalling through FA kinase (FAK). Here, we show that ErbB2 receptor signalling regulates FA turnover, and cell migration and invasion through the Src-FAK pathway. Inhibition of the Src-FAK signalling in ErbB2-positive cells by Herceptin or RNA interference selectively regulates FA turnover, leading to enhanced number and size of peripherally localised adhesions and inhibition of cell invasion. Inhibition of ErbB2 signalling failed to regulate FA and cell migration and invasion in cells lacking FAK or Src but gains this activity after restoration of these proteins. Taken together, our results show a regulation of FA turnover by ErbB2 signalling.

Overexpression of SERTAD3, a putative oncogene located within the 19q13 amplicon, induces E2F activity and promotes tumor growth.

The amplified region of chromosome 19q13.1-13.2 has been associated with several cancers. The well-characterized oncogene AKT2 is located in this amplicon. Two members of the same gene family (SERTAD1 and SERTAD3) are also located within this region. We report herein the genomic structure and potential functions of SERTAD3. SERTAD3 has two transcript variants with short mRNA half-lives, and one of the variants is tightly regulated throughout G1 and S phases of the cell cycle. Overexpression of SERTAD3 induces cell transformation in vitro and tumor formation in mice, whereas inhibition of SERTAD3 by small interfering RNA (siRNA) results in a reduction in cell growth rate. Furthermore, luciferase assays based on E2F-1 binding indicate that SERTAD3 increases the activity of E2F, which is reduced by inhibition of SERTAD3 by siRNA. Together, our data support that SERTAD3 contributes to oncogenesis, at least in part, via an E2F-dependent mechanism.

Protein tyrosine kinase signaling diversity and susceptibility to targeted kinase inhibitors.

Protein kinases, including tyrosine kinases, are one of the largest classes of proteins implicated in cancer development and progression. Recent discovery of selective therapies targeting tyrosine kinase receptor signaling has provided encouraging clinical results. Clinical trials with anti-EGFR, anti-ErbB2/Her2, anti-Bcr-Abl and others have demonstrated the clinical utility of tyrosine kinases as therapeutic targets and as surrogate markers to guide the selection of patients susceptible to respond to treatment. This success has been tempered in part because resistance to targeted therapies is now documented to occur in experimental models and in patients, which hampers therapeutic efficacy. Mechanisms of resistance include cell heterogeneity in target expression, mutations in target's encoding genes, and compensatory signaling mechanisms. This paper provides a brief overview on the diversity of tyrosine kinase signaling and the impact on cancer cell response to targeted tyrosine kinase inhibitors.

Imatinib sensitizes CLL lymphocytes to chlorambucil.

The effect of imatinib on chlorambucil (CLB) cytotoxicity in chronic lymphocytic leukemia (CLL) lymphocytes was examined in vitro. Imatinib sensitizes the WSU and I83 human CLL cell lines, 10- and two-fold, respectively, to CLB. Furthermore, in primary cultures of malignant B-lymphocytes obtained from 12 patients with CLL (seven patients were untreated and five treated with CLB), imatinib synergistically sensitized these lymphocytes from two- to 20-fold to CLB. This synergistic effect was observed at concentrations of imatinib (

Co-administration of finasteride and the pure anti-oestrogen ICI 182,780 act synergistically in modulating the IGF system in rat prostate.

Prostate cancer is the most diagnosed invasive malignancy in males. Androgens and oestrogens have been implicated in the pathogenesis of prostate cancer. We report herein that the pure anti-oestrogen ICI 182,780 (ICI) reduces Ki-67 labelling index and IGF-I receptor levels in rat prostate. Increase of IGF-I mRNA and IGF-binding protein 3 (IGFBP-3) accumulation occur without any effect on prostate weight. Finasteride significantly decreases prostate weight and inhibits IGF-I gene expression. IGFBP-3 mRNA, Akt and phospho-Akt are not affected by finasteride. Co-administration of ICI plus finasteride reduces prostate weight by approximately 50% and causes acinar dilation with decreased luminal epithelial cell thickness. The acinar epithelial cells became atrophic and inactive with minimal cytoplasm. We also demonstrate a synergistic effect of ICI and finasteride on induction of IGFBP-3 accumulation and inhibition of Akt phosphorylation. Because the IGF and IGFBP-3 system plays an important role in prostate epithelial cell proliferation, apoptosis and tumour progression, the inhibitory effects of finasteride and ICI on IGF system may contribute to their anti-proliferative activity. These observations support a potential use of ICI in conjunction with finasteride in the prevention and/or treatment of prostate cancer.

A novel parasite-derived suicide gene for cancer gene therapy with specificity for lung cancer cells.

The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) expressed by the parasite Trypanosoma brucei (Tb) can convert allopurinol, a purine analogue, to corresponding nucleotides with greater efficiency than its human homologue. We have developed a retroviral system that expresses the parasitic enzyme and tested its capacity to activate the prodrug allopurinol to a cytotoxic metabolite. Cytotoxicity assays demonstrated that five non-small cell lung carcinoma cell lines transduced with the construct were sensitized to the prodrug by 2.1- to 7.6-fold compared with control values. This selectivity was not observed in seven other cell lines also expressing the construct, such as breast carcinoma. Assays indicated that enhanced cytotoxicity to allopurinol correlated with induction of apoptosis in lung cancer cells. The selectivity of this suicide gene was not explained either by the TbHGPRT expression or by the allopurinol accumulation. Our study shows that this novel system may represent a therapeutic tool for gene prodrug targeting of lung cancer, considering the fact that allopurinol is well tolerated in humans.

Cloning and characterization of a novel gene that is regulated by estrogen and is associated with mammary gland carcinogenesis.

Estrogens play a role in mammary gland function and are implicated in mammary carcinogenesis. We report the cloning of a novel gene [steroid-sensitive gene 1 (SSG1)] that is regulated by E(2) in the rat uterus and mammary gland. The full-length SSG1 complementary DNA has an open reading frame of 1158 nucleotides encoding a putative protein of 385 amino acids. A SSG1-specific antibody recognizes a 40-kDa protein localized to myoepithelial cells of normal mammary tissue and to endothelial cells of 7,12-dimethylbenz(a)antracene-induced mammary tumors. Treatment of rats with E(2) at 1.2 or 2.4 microg/kg.day for 21 days increases SSG1 protein levels in mammary tissue by 16-fold compared with controls. Removal of E(2) after a 14-day treatment decreases SSG1 protein levels 6-fold and 3-fold at 120 and 144 h, respectively. Treatment of rats with the estrogen antagonists tamoxifen or ICI 182,780 did not affect SSG1 protein levels compared with controls. SSG1 protein levels in 7,12-dimethylbenz(a)antracene-induced rat mammary tumors were 23-fold greater than SSG1 levels in resting mammary tissue, and 8-fold higher than protein levels expressed in lactating mammary glands. We propose that SSG1 plays a role in estrogen functions, and its overexpression is correlated with mammary carcinogenesis.

Steroid-sensitive gene-1 is an androgen-regulated gene expressed in prostatic smooth muscle cells in vivo.

Steroid-sensitive gene-1 (SSG1) is a novel gene we cloned, found regulated by 17beta-estradiol in the rat uterus and mammary gland, and over-expressed in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. We show here that SSG1 mRNA and protein expression are regulated by androgens in the rat ventral prostate. Increases in SSG1 mRNA levels were detected by Northern blotting after 24 h and reached a 27-fold peak 96 h following castration, relative to SSG1 mRNA expression in sham-operated rats. Dihydrotestosterone or testosterone supplementation of castrated rats prevented this rise in SSG1 mRNA. In contrast with SSG1 mRNA expression, SSG1 protein was decreased 16-fold 2 weeks following castration but was at control levels in the prostates of castrated rats receiving dihydrotestosterone or testosterone. Although SSG1 is regulated by androgens in vivo, treatment of LnCap cells with dihydrotestosterone, cyproterone acetate or flutamide did not result in the regulation of SSG1 protein levels in vitro. Immunofluorescence studies show that SSG1 is mainly expressed in prostatic smooth muscle cells. These results indicate that SSG1 is an androgen-regulated gene that is expressed in the smooth muscle component of the rat ventral prostate in vivo.

Action of low calcemic 1alpha,25-dihydroxyvitamin D3 analogue EB1089 in head and neck squamous cell carcinoma.

BACKGROUND: 1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and its analogues inhibit growth of various types of cancer cells. Although the therapeutic potential of 1,25(OH)(2)D(3) is limited by its tendency to induce hypercalcemia, analogues such as EB1089 are potent inhibitors of cell growth and exhibit reduced calcemic effects. We analyzed the antiproliferative and calcemic effects of EB1089 in tissue culture and animal models of head and neck squamous cell carcinoma (SCC) to investigate its potential as a chemotherapeutic/chemopreventive agent. METHODS: The effects of 1,25(OH)(2)D(3) and EB1089 on cell growth and expression of p21(WAF1/CIP1) and p27(KIP1), which encode cyclin-dependent kinase inhibitors, and a novel target, gadd45alpha, a growth-arrest and DNA-damage gene, were monitored in cultured murine AT-84 SCC cells. The effects of these agents on AT-84 cell growth in vitro and on growth of AT-84 tumors in syngeneic C3H mice were monitored; treatment started at the time of tumor implantation (early tumor model) or after 12 days (late tumor model). Weight and serum calcium levels were also monitored in these animals. All P values were two-sided. RESULTS: Both 1,25(OH)(2)D(3) and EB1089 arrested proliferation of AT-84 cells in G(0)/G(1) phase, inhibited p21(WAF1/CIP1) expression, and induced expression of p27(KIP1) protein. 1,25(OH)(2)D(3) also enhanced the expression of gadd45alpha, apparently by a p53-independent mechanism. There was a statistically significant decrease in tumor growth for 1,25(OH)(2)D(3)-treated mice (P<.001 for early tumor model) and EB1089-treated mice (P<.001 and P =.001 for early and late tumor models, respectively). Unlike 1,25(OH)(2)D(3), EB1089 did not induce cachexia or hypercalcemia. The effects of 1,25(OH)(2)D(3) and EB1089 on expression of p21(WAF1/CIP1) and GADD45alpha were similar in tumors and in vitro. CONCLUSIONS: EB1089 completely inhibited growth of AT-84 SCC cells at nanomolar concentrations, reduced tumor growth, and did not have calcemic effects. Our results support continued investigation of EB1089 as a chemopreventive/chemotherapeutic agent for head and neck SCC.

New taxane analogues from the needles of Taxus canadensis.

Eight minor taxanes have been identified for the first time in Taxus canadensis needles. Four of these metabolites are new taxane analogues: 7-acetyl-10-deacetyltaxol (1), 2'-acetyl-7-epi-cephalomannine (2), 10-deacetyl-10-oxo-7-epi-cephalomannine (3), and 10-acetylglycollylbaccatin VI (4).

Transcriptional regulation of the TFIIH transcription repair components XPB and XPD by the hepatitis B virus x protein in liver cells and transgenic liver tissue.

Human hepatitis B virus is a risk factor for the development of hepatocellular carcinoma. The hepatitis B virus x protein (HBx) has been shown to inactivate the p53 tumor suppressor protein and impair DNA repair, cell cycle, and apoptosis mechanisms. Herein we report that HBx represses two components of the transcription-repair factor TFIIH, XPB (p89), and XPD (p80), both in p53-proficient and p53-deficient liver cells. This inhibition is observed while HBx maintains its transactivation function. Expression of HBx in liver cells results in down-regulation of endogenous XPB and XPD mRNAs and proteins; this inhibition is not observed with other TFIIH subunits, XPA or PCNA. In liver tissue from HBx transgenics, XPB and XPD proteins are down-regulated in comparison to matched normal liver tissue. HBx has been shown to interact with Sp1 transcription factor and affects its DNA binding activity. Sp1 is essential for the basal promoter activity of XPB in liver cells and Drosophila SL2 cells. In the Sp1-deficient SL2 cells, HBx-induced XPB and XPD inhibition is Sp1-dependent. In summary, our results provide evidence that HBx represses the expression of key TFIIH proteins at least in part through Sp1 elements; this repression may impair TFIIH function in DNA repair mechanisms.

Upregulation of gap junctional intercellular communication and connexin 43 expression by cyclic-AMP and all-trans-retinoic acid is associated with glutathione depletion and chemosensitivity in neuroblastoma cells.

PURPOSE: Downregulation of gap junctional intercellular communication (GJIC) has been implicated in carcinogenesis. This is a result of altered expression of connexins, the proteins that mediate GJIC, including connexin 43 (Cx43). Our aim was to evaluate the effect of known inducers of Cx43 on the chemosensitivity of the human neuroblastoma cell line IMR-32 to chemotherapeutic agents. METHODS: We examined the effect of dibutyryl-cyclic AMP (db-cAMP) and all-trans-retinoic acid (tRA) on Cx43 and GJIC, glutathione (GSH) and gamma-glutamyl-cysteine-synthetase (gamma-GCS) levels, and glutathione S-transferase (GST) activity. Finally, we performed cell survival assays to measure the response of IMR-32 cells to the chemotherapeutic drugs doxorubicin, melphalan and bis-chloronitrosourea (BCNU), after treatment with db-cAMP and/or tRA. RESULTS: Exposure to db-cAMP led to the upregulation of GJIC and Cx43 expression and phosphorylation. On the other hand, exposure to tRA led to the upregulation of GJIC but Cx43 expression and phosphorylation were not greatly affected. The combination of both agents was more potent in inducing GJIC in comparison to treatment with db-cAMP or tRA alone. Treatment with db-cAMP, but not with tRA, was associated with a significant increase in the cytotoxic effects of the anticancer drugs doxorubicin, melphalan and BCNU as shown by a decrease in their IC50 values. Concomitant exposure to db-cAMP and tRA, however, had a more pronounced effect on cell sensitization to chemotherapy drugs (particularly doxorubicin) than exposure to db-cAMP or tRA alone. Under the db-cAMP and tRA treatment conditions (which upregulate GJIC and modulate drug response), GSH levels were significantly reduced while the levels of GST and gamma-GCS activities remained unchanged. CONCLUSIONS: This study suggests that GJIC plays a role in cellular drug resistance, and highlights the potential use of GJIC modulators in combination with chemotherapy. Also, this is the first study exploring the ability of both db-cAMP and tRA to enhance cell chemosensitivity.

Regulation of the gap junction connexin 43 gene by androgens in the prostate.

Androgens play an important role in prostate gland development and function, and have been implicated in prostate carcinogenesis. We report the regulation of the gap junctional intercellular communication gene connexin 43 (Cx43) by androgens in the prostate gland. In rat ventral prostate tissue, only trace levels of Cx43 mRNA were detected. Castration, however, resulted in a high increase in Cx43 mRNA and protein. Cx32 was unchanged. Castration-induced Cx43 mRNA and protein were abolished by administration of dihydrotestosterone (DHT). Following castration, prostate weights were approximately 16% of sham-treated controls. However, DHT replacement resulted in prostate weights which were not different from sham-treated controls. Under similar castration conditions, Cx43 induction coincided with pronounced apoptosis in the prostate gland cells, and DHT prevented the induction of apoptosis. Given the physiological role of gap junctions and androgens in the regulation of prostate tissue homeostasis, our observations are relevant to the understanding of androgen-dependent prostate carcinogenesis.

RBT1, a novel transcriptional co-activator, binds the second subunit of replication protein A.

Replication Protein A (RPA) is required for DNA recombination, repair and replication in all eukaryotes. RPA participation in these pathways is mediated by single-stranded DNA binding and protein interactions. We herein identify a novel protein, Replication Protein Binding Trans-Activator (RBT1), in a yeast two-hybrid assay employing the second subunit of human RPA (RPA32) as bait. RBT1-RPA32 binding was confirmed by glutathione S:-transferase pull-down and co-immunoprecipitation. Fluorescence microscopy indicates that green fluorescence protein-tagged RBT1 is localized to the nucleus in vivo. RBT1 mRNA expression, determined by semi-quantitative RT-PCR, is significantly higher in cancer cell lines MCF-7, ZR-75, SaOS-2 and H661, compared to the cell lines normal non-immortalized human mammary epithelial cells and normal non-immortalized human bronchial epithelial cells. Further, yeast and mammalian one-hybrid analysis shows that RBT1 is a strong transcriptional co-activator. Interestingly, mammalian transactivation data is indicative of significant variance between cell lines; the GAL4-RBT1 fusion protein has significantly higher transcriptional activity in human cancer cells compared to human normal primary non-immortalized epithelial cells. We propose that RBT1 is a novel transcriptional co-activator that interacts with RPA, and has significantly higher activity in transformed cells.

Modulation of glutathione S-transferase alpha by hepatitis B virus and the chemopreventive drug oltipraz.

Persistent infection by hepatitis B virus (HBV) and exposure to chemical carcinogens correlates with the prevalence of hepatocellular carcinoma in endemic areas. The precise nature of the interaction between these factors is not known. Glutathione S-transferases (GST) are responsible for the cellular metabolism and detoxification of a variety of cytotoxic and carcinogenic compounds by catalysis of their conjugation with glutathione. Diminished GST activity could enhance cellular sensitivity to chemical carcinogens. We have investigated GST isozyme expression in hepatocellular HepG2 cells and in an HBV-transfected subline. Total GST activity and selenium-independent glutathione peroxidase activity are significantly decreased in HBV transfected cells. On immunoblotting, HBV transfected cells demonstrate a significant decrease in the level of GST Alpha class. Cytotoxicity assays reveal that the HBV transfected cells are more sensitive to a wide range of compounds known to be detoxified by GST Alpha conjugation. Although no significant difference in protein half-life between the two cell lines was found, semi-quantitative reverse transcription-polymerase chain reaction shows a reduced amount of GST Alpha mRNA in the transfected cells. Because the HBV x protein (HBx) seems to play a role in HBV transfection, we also demonstrated that expression of the HBx gene into HepG2 cells decreased the amount of GST Alpha protein. Transient transfection experiments using both rat and human GST Alpha (rGSTA5 and hGSTA1) promoters in HepG2 cells show a decreased CAT activity upon HBx expression, supporting a transcriptional regulation of both genes by HBx. This effect is independent of HBx interaction with Sp1. Treatment with oltipraz, an inducer of GST Alpha, partially overcomes the effect of HBx on both promoters. Promoter deletion studies indicate that oltipraz works through responsive elements distinct from AP1 or NF-kappaB transcription factors. Thus, HBV infection alters phase II metabolizing enzymes via different mechanisms than those modulated by treatment with oltipraz.

Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis.

The interaction between the erbB tyrosine kinase receptors and their ligands plays an important role in tumor growth via the regulation of autocrine and paracrine loops. We report the effect of heregulin beta1, the ligand for erbB-3 and erbB-4 receptors, on the regulation of vascular endothelial growth factor (VEGF) expression, using a panel of breast and lung cancer cell lines with constitutive erbB-2 overexpression or engineered to stably overexpress the erbB-2 receptor. We demonstrate that heregulin beta1 induces VEGF secretion in most cancer cell lines, while no significant effect was observed in normal human mammary and bronchial primary cells. Overexpression of erbB-2 receptor results in induction of the basal level of VEGF and exposure to heregulin further enhances VEGF secretion. This is associated with increased VEGF mRNA expression. In contrast, VEGF induction is significantly decreased in a T47D cell line where erbB-2 is functionally inactivated. Conditioned media from heregulin-treated cancer cells, but not from normal cells, stimulates endothelial cell proliferation; this paracrine stimulation is inhibited by co-exposure to a specific VEGF neutralizing antibody. Furthermore, heregulin-mediated angiogenesis is observed in the in vivo CAM assay. This study reports the first evidence of VEGF regulation by heregulin in cancer cells. Oncogene (2000) 19, 3460 - 3469

Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis.

Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8, 9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress.

Identification of the interferon-inducible double-stranded RNA-dependent protein kinase as a regulator of cellular response to bulky adducts.

The double-stranded RNA-dependent protein kinase PKR plays a central role in IFN-mediated antiviral response. The ability of PKR mutants to transform rodent fibroblasts led to the hypothesis that PKR acts as a tumor suppressor. Recent studies have identified an expanding network of PKR signaling partners, including signal transducers and activators of transcription 1 (STAT1), p53, and IkappaB-kinase. Here we demonstrate that PKR is involved in the cellular response to genotoxic stress. PKR-deficient mouse-embryonic fibroblasts (PKR-/-) are hypersensitive to bulky adduct DNA damage caused by cisplatin, melphalan, and UV radiation but not to other DNA-damaging agents such as Adriamycin. PKR-deficient cells are highly susceptible to cisplatin-induced apoptosis. They demonstrate retarded cisplatin adduct removal kinetics. Most strikingly, PKR localizes to the nucleus rapidly upon cisplatin treatment. Restoration of PKR in PKR-/- cells results in resistance to cisplatin and enhanced cell capacity to remove cisplatin DNA adducts. We conclude that PKR has a function in the regulation of cellular response to bulky adduct-inducing agents, possibly by modulating DNA repair mechanisms.