Harnessing and Optimizing the Interplay between Immunotherapy and Radiotherapy to Improve Survival Outcomes.

In the past, radiotherapy was primarily used to control local disease, but recent technological advances in accurate, high-dose ionizing radiation (IR) delivery have not only increased local tumor control but in some cases reduced metastatic burden. These "off target" therapeutic effects of IR at nonirradiated tumor sites, also known as abscopal effects, are thought to be mediated by tumor antigen-primed T cells that travel to metastatic sites and promote tumor regression. Similarly, early indications reveal that IR in combination with immune checkpoint inhibitors, such as ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1), can provide superior therapeutic responses. These observations suggest that local radiotherapy results in altered gene expression, exposure of new antigens, or cell death that can interact with immunotherapy. As such, radiotherapy enhancement of immune responses offers a promising synergy with the potential for substantial clinical benefit. This review focuses on the biology that underlies the mechanisms for the interaction between radiation-induced tumor cell death and enhanced immunologic response. Mol Cancer Res; 16(8); 1209-14. ©2018 AACR.

Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination.

The nitric oxide (NO)-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR) to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR) repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1). Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18), which causes stem cell differentiation has no effect on double-strand break (DSB) repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.

MCL-1 Depletion Impairs DNA Double-Strand Break Repair and Reinitiation of Stalled DNA Replication Forks.

Myeloid cell leukemia 1 (MCL-1) is a prosurvival BCL-2 protein family member highly expressed in hematopoietic stem cells (HSCs) and regulated by growth factor signals that manifest antiapoptotic activity. Here we report that depletion of MCL-1 but not its isoform MCL-1S increases genomic instability and cell sensitivity to ionizing radiation (IR)-induced death. MCL-1 association with genomic DNA increased postirradiation, and the protein colocalized with 53BP1 foci. Postirradiation, MCL-1-depleted cells exhibited decreased γ-H2AX foci, decreased phosphorylation of ATR, and higher levels of residual 53BP1 and RIF1 foci, suggesting that DNA double-strand break (DSB) repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1-depleted cells had a reduced frequency of IR-induced BRCA1, RPA, and Rad51 focus formation, decreased DNA end resection, and decreased HR repair in the DR-GFP DSB repair model. Similarly, after HU induction of stalled replication forks in MCL-1-depleted cells, there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR-mediated resolution of collapsed forks. Therefore, the present data support a model whereby MCL-1 depletion increases 53BP1 and RIF1 colocalization at DSBs, which inhibits BRCA1 recruitment, and sensitizes cells to DSBs from IR or stalled replication forks that require HR for repair.

A multifaceted role for MOF histone modifying factor in genome maintenance.

MOF (males absent on the first) was initially identified as a dosage compensation factor in Drosophila that acetylates lysine 16 of histone H4 (H4K16ac) and increased gene transcription from the single copy male X-chromosome. In humans, however, the ortholog of Drosophila MOF has been shown to interact with a range of proteins that extend its potential significance well beyond transcription. For example, recent results indicate MOF is an upstream regulator of the ATM (ataxia-telangiectasia mutated) protein, the loss of which is responsible for ataxia telangiectasia (AT). ATM is a key regulatory kinase that interacts with and phosphorylates multiple substrates that influence critical, cell-cycle control and DNA damage repair pathways in addition to other pathways. Thus, directly or indirectly, MOF may be involved in a wide range of cellular functions. This review will focus on the contribution of MOF to cellular DNA repair and new results that are beginning to examine the in vivo physiological role of MOF.

Novel association of DJ-1 with HER3 potentiates HER3 activation and signaling in cancer.

HER3/ErbB3 has emerged as a new therapeutic target for cancer. Currently, more than a dozen anti-HER3 antibodies are in clinical trials for treatment of various cancers. However, limited understanding of the complex HER3 signaling in cancer and lack of established biomarkers have made it challenging to stratify cancer patients who can benefit from HER3 targeted therapies. In this study, we identified DJ-1/PARK7 (Parkinson Protein 7) as a novel interaction partner of HER3 and demonstrated the potential of DJ-1 as a biomarker for anti-HER3 cancer therapy. DJ-1 association with HER3 protects HER3 from ubiquitination and degradation through the proteasomal pathway in breast cancer cells. However, neuregulin 1 (NRG-1) mediated HER3 activation results in a reduced association of DJ-1 with HER3. DJ-1 shRNA knockdown in cancer cells resulted in decreased levels of HER3 and its downstream signaling through the PI3K/AKT and Ras/Raf/ERK pathways. DJ-1 shRNA knockdown cancer cells significantly reduced cell proliferation and migration in vitro and tumor growth in vivo. Conversely, overexpression of DJ-1 increased HER3 levels and promoted cancer cell proliferation in vitro and tumor growth in vivo. Notably, cancer cells with high DJ-1 expression showed more sensitivity than DJ-1 knockdown cells to anti-HER3 antibody inhibition. In addition, there was a significant co-expression of HER3 and DJ-1 in tumor tissues of breast cancer patients. Taken together, these results suggest that high DJ-1 expression in breast cancer cells predicts elevated HER3 signaling and may therefore serve as a biomarker for HER3 targeted antibody cancer therapies.

Pluripotent Stem Cells and DNA Damage Response to Ionizing Radiations.

Pluripotent stem cells (PSCs) hold great promise in regenerative medicine, disease modeling, functional genomics, toxicological studies and cell-based therapeutics due to their unique characteristics of self-renewal and pluripotency. Novel methods for generation of pluripotent stem cells and their differentiation to the specialized cell types such as neuronal cells, myocardial cells, hepatocytes and beta cells of the pancreas and many other cells of the body are constantly being refined. Pluripotent stem cell derived differentiated cells, including neuronal cells or cardiac cells, are ideal for stem cell transplantation as autologous or allogeneic cells from healthy donors due to their minimal risk of rejection. Radiation-induced DNA damage, ultraviolet light, genotoxic stress and other intrinsic and extrinsic factors triggers a series of biochemical reactions known as DNA damage response. To maintain genomic stability and avoid transmission of mutations into progenitors cells, stem cells have robust DNA damage response signaling, a contrast to somatic cells. Stem cell transplantation may protect against radiation-induced late effects. In particular, this review focuses on differential DNA damage response between stem cells and derived differentiated cells and the possible pathways that determine such differences.

ß2-spectrin depletion impairs DNA damage repair.

ß2-Spectrin (ß2SP/SPTBN1, gene SPTBN1) is a key TGF-ß/SMAD3/4 adaptor and transcriptional cofactor that regulates TGF-ß signaling and can contribute to liver cancer development. Here we report that cells deficient in ß2-Spectrin (ß2SP) are moderately sensitive to ionizing radiation (IR) and extremely sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with IR or ICL agents (formaldehyde, cisplatin, camptothecin, mitomycin), ß2SP deficient cells displayed a higher frequency of cells with delayed γ-H2AX removal and a higher frequency of residual chromosome aberrations. Following hydroxyurea (HU)-induced replication stress, ß2SP-deficient cells displayed delayed disappearance of γ-H2AX foci along with defective repair factor recruitment (MRE11, CtIP, RAD51, RPA, and FANCD2) as well as defective restart of stalled replication forks. Repair factor recruitment is a prerequisite for initiation of DNA damage repair by the homologous recombination (HR) pathway, which was also defective in ß2SP deficient cells. We propose that ß2SP is required for maintaining genomic stability following replication fork stalling, whether induced by either ICL damage or replicative stress, by facilitating fork regression as well as DNA damage repair by homologous recombination.

Crk II silencing down-regulates IGF-IR and inhibits migration and invasion of prostate cancer cells.

Crk (C10 regulator of kinase) adaptor proteins are highly expressed in many types of human cancers and often contribute to aggressive cancer phenotypes. Crk II, a member of CRK family, has been reported to regulate cell migration and metastasis in breast cancer cells. However, its role in other cancer types has not been reported. In this study, we investigated the molecular function of Crk II in prostate cancer (PCa) cells (CWR-22rv1) in vitro and using a mouse tumor model. Results showed that Crk II knockdown by shRNA-mediated silencing (Crk II-shRNA) in the PCa cells significantly inhibited both cancer cell migration and invasion in cell culture study. Crk II-shRNA cancer cells also significantly decreased colony formation in vitro, but had no significant reduction of tumor volume after 4 weeks of cancer cell xenografting in vivo when compared to the scramble control. Interestingly, Crk II-shRNA cancer cells showed a greatly reduced level of insulin-like growth factor 1 receptor (IGF-1R) and decreased signaling of the IGF-1R/PI3K/Akt axis upon IGF-1 ligand stimulation. A close interaction between Crk II and IGF-1R was demonstrated upon co-immunoprecipitation of IGF-1R with Crk II protein. Further, treatment of cells with either proteosomal degradation or protein synthesis inhibitor showed higher proportion of ubiquitin-associated IGF-1R and faster degradation of IGF-1R in Crk II-shRNA cells in comparison with that in the control cancer cells. Taken together, these data suggest that Crk II plays an important role in the regulation of IGF-1R protein stability and affects downstream of IGF-1R signaling pathways. Therefore, targeting Crk-II can block IGF-1R growth signaling and suppress cancer cell invasion and progression.

Role of the Exocyst Complex Component Sec6/8 in Genomic Stability.

The exocyst is a heterooctomeric complex well appreciated for its role in the dynamic assembly of specialized membrane domains. Accumulating evidence indicates that this macromolecular machine also serves as a physical platform that coordinates regulatory cascades supporting biological systems such as host defense signaling, cell fate, and energy homeostasis. The isolation of multiple components of the DNA damage response (DDR) as exocyst-interacting proteins, together with the identification of Sec8 as a suppressor of the p53 response, suggested functional interactions between the exocyst and the DDR. We found that exocyst perturbation resulted in resistance to ionizing radiation (IR) and accelerated resolution of DNA damage. This occurred at the expense of genomic integrity, as enhanced recombination frequencies correlated with the accumulation of aberrant chromatid exchanges. Sec8 perturbation resulted in the accumulation of ATF2 and RNF20 and the promiscuous accumulation of DDR-associated chromatin marks and Rad51 repairosomes. Thus, the exocyst supports DNA repair fidelity by limiting the formation of repair chromatin in the absence of DNA damage.

Regulation of ERBB3/HER3 signaling in cancer.

ERBB3/HER3 is emerging as a molecular target for various cancers. HER3 is overexpressed and activated in a number of cancer types under the conditions of acquired resistance to other HER family therapeutic interventions such as tyrosine kinase inhibitors and antibody therapies. Regulation of the HER3 expression and signaling involves numerous HER3 interacting proteins. These proteins include PI3K, Shc, and E3 ubiquitin ligases NEDD4 and Nrdp1. Furthermore, recent identification of a number of HER3 oncogenic mutations in colon and gastric cancers elucidate the role of HER3 in cancer development. Despite the strong evidence regarding the role of HER3 in cancer, the current understanding of the regulation of HER3 expression and activation requires additional research. Moreover, the lack of biomarkers for HER3-driven cancer poses a big challenge for the clinical development of HER3 targeting antibodies. Therefore, a better understanding of HER3 regulation should improve the strategies to therapeutically target HER3 for cancer therapy.

Curcumin induces differentiation of embryonic stem cells through possible modulation of nitric oxide-cyclic GMP pathway.

Curcumin, an active ingredient of dietary spice used in curry, has been shown to exhibit anti-oxidant, anti-inflammatory and anti-proliferative properties. Using EB directed differentiation protocol of H-9 human embryonic stem (ES) cells; we evaluated the effect of curcumin (0-20 µmol/L) in enhancing such differentiation. Our results using real time PCR, western blotting and immunostaining demonstrated that curcumin significantly increased the gene expression and protein levels of cardiac specific transcription factor NKx2.5, cardiac troponin I, myosin heavy chain, and endothelial nitric oxide synthase during ES cell differentiation. Furthermore, an NO donor enhanced the curcumin-mediated induction of NKx2.5 and other cardiac specific proteins. Incubation of cells with curcumin led to a dose dependent increase in intracellular nitrite to the same extent as giving an authentic NO donor. Functional assay for second messenger(s) cyclic AMP (cAMP) and cyclic GMP (cGMP) revealed that continuous presence of curcumin in differentiated cells induced a decrease in the baseline levels of cAMP but it significantly elevated baseline contents of cGMP. Curcumin addition to a cell free assay significantly suppressed cAMP and cGMP degradation in the extracts while long term treatment of intact cells with curcumin increased the rates of cAMP and cGMP degradation suggesting that this might be due to direct suppression of some cyclic nucleotide-degrading enzyme (phosphodiesterase) by curcumin. These studies demonstrate that polyphenol curcumin may be involved in differentiation of ES cells partly due to manipulation of nitric oxide signaling.

Nitric oxide-cyclic GMP signaling in stem cell differentiation.

The nitric oxide-cyclic GMP (NO-cGMP) pathway mediates important physiological functions associated with various integrative body systems including the cardiovascular and nervous systems. Furthermore, NO regulates cell growth, survival, apoptosis, proliferation, and differentiation at the cellular level. To understand the significance of the NO-cGMP pathway in development and differentiation, studies have been conducted both in developing embryos and in stem cells. Manipulation of the NO-cGMP pathway, by employing activators and inhibitors as pharmacological probes, and genetic manipulation of NO signaling components have implicated the involvement of this pathway in the regulation of stem cell differentiation. This review focuses on some of the work pertaining to the role of NO-cGMP in the differentiation of stem cells into cells of various lineages, particularly into myocardial cells, and in stem cell-based therapy.

Nitric oxide receptor soluble guanylyl cyclase undergoes splicing regulation in differentiating human embryonic cells.

Nitric oxide (NO), an important mediator molecule in mammalian physiology, initiates a number of signaling mechanisms by activating the enzyme soluble guanylyl cyclase (sGC). Recently, a new role for NO/cyclic guanosine monophosphate signaling in embryonic development and cell differentiation has emerged. The changes in expression of NO synthase isoforms and various sGC subunits has been demonstrated during human and mouse embryonic stem (ES) cells differentiation. Previously, our laboratory demonstrated that nascent α1 sGC transcript undergoes alternative splicing and that expression of α1 sGC splice forms directly affects sGC activity. Expression of sGC splice variants in the process of human ES (hES) cells differentiation has not been investigated. In this report, we demonstrate that α1 sGC undergoes alternative splicing during random hES differentiation for the first time. Our results indicate that C-α1 sGC splice form is expressed at high levels in differentiating cells and its intracellular distribution varies from canonical α1 sGC subunit. Together, our data suggest that alternative splicing of sGC subunits is associated with differentiation of hES cells.

Role of soluble guanylyl cyclase-cyclic GMP signaling in tumor cell proliferation.

Our previous studies demonstrate a differential expression of nitric oxide (NO) signaling components in ES cells and our recent study demonstrated an enhanced differentiation of ES cells into myocardial cells with NO donors and soluble guanylyl cyclase (sGC) activators. Since NO-cGMP pathway exhibits a diverse role in cancer, we were interested in evaluating the role of the NO-receptor sGC and other components of the pathway in regulation of the tumor cell proliferation. Our results demonstrate a differential expression of the sGC subunits, NOS-1 and PKG mRNA and protein levels in various human cancer models. In contrast to sGC alpha(1), robust levels of sGC beta(1) were observed in OVCAR-3 (ovarian) and MDA-MB-468 (breast) cancer cells which correlated well with the sGC activity and a marked increase in cGMP levels upon exposure to the combination of a NO donor and a sGC activator. NOC-18 (DETA NONOate; NO donor), BAY41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine); sGC activator), NOC-18+BAY41-2272, IBMX (3-isobutyl-1-methylxanthine; phosphodiesterase inhibitor) and 8-bromo-cGMP (cGMP analog) caused growth inhibition and apoptosis in various cancer cell lines. To elucidate the molecular mechanisms involved in growth inhibition, we evaluated the effect of activators/inhibitors on ERK phosphorylation. Our studies indicate that BAY41-2272 or the combination NOC-18+BAY41-2272 caused inhibition of the basal ERK1/2 phosphorylation in OVCAR-3 (high sGC activity), SK-OV-3 and SK-Br-3 (low sGC activity) cell lines and in some cases the inhibition was rescued by the sGC inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). These studies suggest that the effects of activators/inhibitors of NO-sGC-cGMP in tumor cell proliferation is mediated by both cGMP-dependent and independent mechanisms.

Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells.

Nitric oxide (NO) is involved in number of physiological and pathological events. Our previous studies demonstrated a differential expression of NO signaling components in mouse and human ES cells. Here, we demonstrate the effect of NO donors and soluble guanylyl cyclase (sGC) activators in differentiation of ES cells into myocardial cells. Our results with mouse and human ES cells demonstrate an increase in Nkx2.5 and myosin light chain (MLC2) mRNA expression on exposure of cells to NO donors and a decrease in mRNA expression of both cardiac-specific genes with nonspecific NOS inhibitor and a concomitant increase and decrease in the mRNA levels of sGC alpha(1) subunit. Although sGC activators alone exhibited an increase in mRNA expression of cardiac genes (MLC2 and Nkx2.5), robust inductions of mRNA and protein expression of marker genes were observed when NO donors and sGC activators were combined. Measurement of NO metabolites revealed an increase in the nitrite levels in the conditioned media and cell lysates on exposure of cells to the different concentrations of NO donors. cGMP analysis in undifferentiated stem cells revealed a lack of stimulation with NO donors. Differentiated cells however, acquired the ability to be stimulated by NO donors. Although, 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b]pyridine (BAY 41-2272) alone was able to stimulate cGMP accumulation, the combination of NO donors and BAY 41-2272 stimulated cGMP levels more than either of the agents separately. These studies demonstrate that cGMP-mediated NO signaling plays an important role in the differentiation of ES cells into myocardial cells.

Differential expression of nitric oxide signaling components in undifferentiated and differentiated human embryonic stem cells.

Nitric oxide (NO) is an uncharged free-radical gas that is involved in a number of physiological and pathological events. We have examined the expression of various subunits of soluble guanylyl cyclase (sGC alpha (1), alpha (2), beta (1), beta (2)), nitric oxide synthase (s) (NOS-1, -2, -3), MLC2 (cardiac marker) and a cardiac-specific transcription factor (Nkx2.5) in human embryonic stem (hES) cells (H-9 cells) and differentiated cells subjected to differentiation in cell suspension using embryoid body (EB) formation. Our results demonstrate a time-dependent increase in the expression of sGC alpha (1) and beta (1) at the mRNA and protein levels in differentiated cells compared to undifferentiated H-9 cells as examined by real-time PCR and western blot analysis. mRNA for sGC alpha (2) also showed a time-dependent increase compared to undifferentiated cells. In contrast, there was a time-dependent decrease in sGC beta (2) mRNA expression in differentiated cells compared to undifferentiated H-9 cells. In contrast to undifferentiated H-9 cells, the maximum mRNA expression of cardiac marker MLC2 and the cardiac-specific transcription factor Nkx2.5 was observed at day 14 of the differentiated H-9 cells. The protein levels of MLC2 were stable up to day 25 compared to mRNA levels, which showed a sharp decline after day 15. Using immunofluorescence, we also demonstrate positive staining of cardiac markers such as troponin I, alpha-actinin, atrial natriuretic peptide, and SGC alpha (1) at days 8-37 post-differentiation. These results clearly demonstrate the role of NO signaling components in differentiation events or physiological processes of human ES or ES cell-derived cardiomyocytes.

Increased sensitivity of a metastatic model of prostate cancer to a novel tetravalent platinum analog.

BACKGROUND: DACH-Ac-Pt [(1R,2R-diaminocyclohexane)-(trans-diacetato)-(dichloro)-platinum(IV)] is a novel cisplatin (CDDP) analog, and we have evaluated its potential activity in human prostate cancers. METHODS: Cytotoxic, biochemical pharmacologic, cell cycle, and Western blot evaluations were conducted with platinum agents to assess the role of p53 genotype and androgen-dependence status on cellular response. RESULTS: CDDP and DACH-Ac-Pt were equiactive against mutant p53 and androgen-independent DU-145 or PC-3 tumor cells. In wild-type p53 cells, CDDP was threefold more potent against androgen-dependent LNCaP than isogenic androgen-independent LNCaP-LN3 cells. However, the analog was equipotent in these two wild-type p53 tumor models. The greater potency of DACH-Ac-Pt than CDDP in wild-type p53 cells was not due to increased cellular drug uptake or increased adduct levels, but correlated with a lower tolerance to DNA damage. The analog also activated the p53-p21(WAF1/CIP1) signal transduction pathway more efficiently in LNCaP and LNCaP-LN3 cells, and this induced G(1)-phase cell-cycle arrest. CDDP, in contrast, activated this pathway efficiently in LNCaP cells only. In addition, and compared to CDDP, DACH-Ac-Pt was more effective in inducing Bax and increasing the Bax/Bcl-2 ratios in both the tumor models. CONCLUSIONS: DACH-Ac-Pt is highly effective against wild-type p53 LNCaP and its LN3 variant, and this activity is androgen-independent. The differential induction of p21(WAF1/CIP1) and increase in Bax/Bcl-2 ratios with CDDP and DACH-Ac-Pt in LNCaP-LN3 cells appear to be linked to the relative activity of the two agents against this model.

Status of p53 phosphorylation and function in sensitive and resistant human cancer models exposed to platinum-based DNA damaging agents.

PURPOSE: Resistance to chemotherapeutic drugs is a hallmark of many human cancers, which can occur independent of p53 gene status; however, the presence of wild-type p53 in chemorefractory tumors confers greater resistance to cisplatin, but such tumors do not display complete cross-resistance to the platinum analog (1R,2R-diaminocyclohexane)(trans-diacetato)(dichloro)platinumIV (DACH-Ac-Pt). In this article we examine DNA damage-induced phosphorylation of p53 and downstream p53-dependent transactivation events in cisplatin-sensitive and cisplatin-resistant human cancer cell lines possessing wild-type p53. METHODS: Western-blot analysis was utilized to study the effect of cisplatin and the analog on p53 phosphorylation and p53-dependent target genes. RESULTS: In response to CDDP and DACH-Ac-Pt, both CDDP-sensitive and CDDP-resistant models demonstrated time- and dose-dependent inductions of total p53 protein and an increase in Ser-15 phosphorylation, which was more pronounced with CDDP. Although phosphorylation of p53 at Ser-392 was also observed in CDDP-treated sensitive and resistant cells, it was weak or absent in response to DACH-Ac-Pt. Lack of Ser-392 phosphorylation by DACH-Ac-Pt, however, did not affect the induction of p21(WAF1/CIP1) or Mdm2. Similarly, inductions of p21(WAF1/CIP1) and Mdm2 were observed in sensitive cells exposed to cisplatin. In marked contrast, cisplatin-mediated induction of p21(WAF1/CIP1) was minimal or absent in resistant cells, but that of Mdm2 was unaffected. Wortmannin, a PI3-kinase (PI3-K) inhibitor, caused a dose-dependent inhibition of total p53 accumulation, Ser-15 phosphorylation and p21(WAF1/CIP1) transactivation in response to both CDDP and DACH-Ac-Pt, indicating that members of the PI3-K family are involved in phosphorylation of p53 and that transactivation of p21(WAF1/CIP1) is p53 dependent. CONCLUSION: These studies demonstrate that cisplatin and DACH-Ac-Pt differentially phosphorylate p53 through independent DNA damage-induced pathways, and that the kinase-mediated phosphorylation of p53 at Ser-15 or Ser-392 is unaltered in resistance. Moreover, the phosphorylation status of Ser-392 on its own does not appear to correlate with p21(WAF1/CIP1) or Mdm2 induction in these studies; however, a lack of increase in p21(WAF1/CIP1) by cisplatin, but not DACH-Ac-Pt, provides a correlation with resistance and its circumvention, and implicates the role for cyclin-dependent kinase inhibitor in the differential cytotoxic effects of the two platinum agents against resistant cells.