Deletions of CDKN2A and MTAP Detected by Copy-Number Variation Array Are Associated with Loss of p16 and MTAP Protein in Pleural Mesothelioma.

CDKN2A deletion is a common alteration in pleural mesothelioma (PM) and frequently associated with co-deletion of MTAP. Since the standard detection method for CDKN2A deletion and FISH analysis is relatively expensive, we here investigated the suitability of inexpensive p16 and MTAP IHC by comparing concordance between IHC and OncoScan CNV arrays on samples from 52 PM patients. Concordance was determined using Cohen's kappa statistics. Loss of CDKN2A was associated with co-deletion of MTAP in 71% of cases. CDKN2A-MTAP copy-number normal cases were also IHC positive in 93% of cases for p16 and 100% for MTAP, while homozygous deletion of CDKN2A-MTAP was always associated with negative IHC for both proteins. In cases with heterozygous CDKN2A-MTAP loss, IHC expression of p16 and MTAP was negative in 100% and 71%, respectively. MTAP and p16 IHC showed high sensitivity (MTAP 86.5%, p16 100%) and specificity (MTAP 100%, p16 93.3%) for the detection of any gene loss. Loss of MTAP expression occurred exclusively in conjunction with loss of p16 labeling. Both p16 and MTAP IHC showed high concordance with Oncoscan CNV arrays (kappa = 0.952, p < 0.0001, and kappa = 0.787, p < 0.0001 respectively). We recommend combined MTAP and p16 immunohistochemistry to confirm the diagnosis of PM.

Cullin 4B Ubiquitin Ligase Is Important for Cell Survival and Regulates TGF-ß1 Expression in Pleural Mesothelioma.

We previously demonstrated that cullin 4B (CUL4B) upregulation was associated with worse outcomes of pleural mesothelioma (PM) patients, while the overexpression of its paralog CUL4A was not associated with clinical outcomes. Here, we aimed to identify the distinct roles of CUL4B and CUL4A in PM using an siRNA approach in PM cell lines (ACC Meso-1 and Mero82) and primary culture. The knockdown of CUL4B and CUL4A resulted in significantly reduced colony formation, increased cell death, and delayed cell proliferation. Furthermore, similar to the effect of CUL4A knockdown, downregulation of CUL4B led to reduced expression of Hippo pathway genes including YAP1, CTGF, and survivin. Interestingly, CUL4B and not CUL4A knockdown reduced TGF-ß1 and MMP2 expression, suggesting a unique association of CUL4B with this pathway. However, the treatment of PM cells with exogenous TGF-ß1 following CUL4B knockdown did not rescue PM cell growth. We further analyzed ACC Meso-1 xenograft tumor tissues treated with the cullin inhibitor, pevonedistat, which targets protein neddylation, and observed the downregulation of human TGF-ß1 and MMP2. In summary, our data suggest that CUL4B overexpression is important for tumor cell growth and survival and may drive PM aggressiveness via the regulation of TGF-ß1 expression and, furthermore, reveal a new mechanism of action of pevonedistat.

Alterations in BAP1 Are Associated with Cisplatin Resistance through Inhibition of Apoptosis in Malignant Pleural Mesothelioma.

PURPOSE: The clinical standard treatment for patients with malignant pleural mesothelioma (MPM) includes a cisplatin-based chemotherapy, leading to reduction of tumor size in only a minority of patients. Predicting response to chemotherapy in patients with MPM by using a genetic marker would, therefore, enable patient stratification. EXPERIMENTAL DESIGN: In this retrospective biomarker study, eligible patients had resectable MPM, measurable disease, and available primary MPM tissue. All patients underwent first-line treatment with cisplatin and pemetrexed, followed by surgery. Thorough molecular analysis was performed (whole-exome and targeted deep sequencing, and copy-number analyses), and also mechanistic in vitro data (viability assays, Western blots, and immunoprecipitation) using mesothelioma cell lines with and without siRNA-mediated BRCA1-associated protein 1 (BAP1) knockdown were provided. RESULTS: In a training cohort of patients with MPM (n = 28), mutations or deletions of BAP1 each predicted resistance to chemotherapy in patients with primary MPM. The negative predictive value of BAP1 loss in patients with MPM was confirmed by amplicon sequencing and copy-number array technology in an independent test cohort (n = 39). Preliminary mechanistic studies using siRNA-based knockdown of BAP1 in MPM cell culture models along with immunoprecipitation assays confirmed chemoresistance in vitro, possibly through inhibition of apoptosis and transcriptional regulation of the BAP1/HCF1/E2F1 axis. CONCLUSIONS: Alterations in BAP1 in MPM were a negative predictor for response to chemotherapy and could possibly be used as a companion biomarker for treatment decision.

Importance of Cullin4 Ubiquitin Ligase in Malignant Pleural Mesothelioma.

Neurofibromatosis type 2 (NF2), the tumor suppressor frequently lost in malignant pleural mesothelioma (MPM), suppresses tumorigenesis in part by inhibiting the Cullin4 ubiquitin ligase (CUL4) complex in the nucleus. Here, we evaluated the importance of CUL4 in MPM progression and tested the efficacy of cullin inhibition by pevonedistat, a small molecule inhibiting cullin neddylation. CUL4 paralogs (CUL4A and CUL4B) were upregulated in MPM tumor specimens compared to nonmalignant pleural tissues. High gene and protein expressions of CUL4B was associated with a worse progression-free survival of MPM patients. Among 13 MPM cell lines tested, five (38%) were highly sensitive to pevonedistat (half maximal inhibitory concentration of cell survival IC50 < 0.5 µM). This remained true in a 3D spheroid culture. Pevonedistat treatment caused the accumulation of CDT1 and p21 in both sensitive and resistant cell lines. However, the treatment induced S/G2 cell cycle arrest and DNA rereplication predominantly in the sensitive cell lines. In an in vivo mouse model, the pevonedistat treatment significantly prolonged the survival of mice bearing both sensitive and resistant MPM tumors. Pevonedistat treatment reduced growth in sensitive tumors but increased apoptosis in resistant tumors. The mechanism in the resistant tumor model may be mediated by reduced macrophage infiltration, resulting from the suppression of macrophage chemotactic cytokines, C-C motif chemokine ligand 2 (CCL2), expression in tumor cells.

Cytosolic pH regulates proliferation and tumour growth by promoting expression of cyclin D1.

Enhanced growth and proliferation of cancer cells are accompanied by profound changes in cellular metabolism. These metabolic changes are also common under physiological conditions, and include increased glucose fermentation accompanied by elevated cytosolic pH (pHc)1,2. However, how these changes contribute to enhanced cell growth and proliferation is unclear. Here, we show that elevated pHc specifically orchestrates an E2F-dependent transcriptional programme to drive cell proliferation by promoting cyclin D1 expression. pHc-dependent transcription of cyclin D1 requires the transcription factors CREB1, ATF1 and ETS1, and the histone acetyltransferases p300 and CBP. Biochemical characterization revealed that the CREB1-p300/CBP interaction acts as a pH sensor and coincidence detector, integrating different mitotic signals to regulate cyclin D1 transcription. We also show that elevated pHc contributes to increased cyclin D1 expression in malignant pleural mesotheliomas (MPMs), and renders these cells hypersensitive to pharmacological reduction of pHc. Taken together, these data demonstrate that elevated pHc is a critical cellular signal regulating G1 progression, and provide a mechanism linking elevated pHc to oncogenic activation of cyclin D1 in MPMs, and possibly other cyclin D1~dependent tumours. Thus, an increase of pHc may represent a functionally important, early event in the aetiology of cancer that is amenable to therapeutic intervention.

Intracavitary cisplatin-fibrin chemotherapy after surgery for malignant pleural mesothelioma: A phase I trial.

OBJECTIVES: Intracavitary chemotherapy is a promising concept to improve local tumor control for malignant pleural mesothelioma with reported high morbidity rates. We have demonstrated that administration of cisplatin loaded to fibrin increased local drug concentration and reduced systemic toxicity in preclinical models. We present a phase I trial of intracavitary cisplatin-fibrin after surgical tumor resection. METHODS: A total of 12 patients (75% International Mesothelioma Interest Group stage III-IV) were treated with 4 dose levels of intracavitary cisplatin-fibrin (11-44 mg/m2 body surface area) in a dose-escalating design. Cisplatin-fibrin was sprayed on the resected surfaces after pleurectomy/decortication. Blood and tissue samples were taken to assess toxicity and pharmacokinetics. Patients were regularly followed up. RESULTS: No dose-limiting toxicity was observed. Major morbidity occurred in 4 patients (33%). The 30-day and 90-day mortality were both 0%. Of 80 adverse events, 9 were classified serious, but none of these were related to study treatment. Local cisplatin concentration in the chest wall tissue was high at all dose levels (median, 46.3 µg/g [12-133 µg/g]). In serum, median cisplatin area under the concentration time curve values were always below renal toxicity levels. The median overall survival with 95% confidence interval was 21 months (10-31 months). In 1 patient with epithelioid malignant pleural mesothelioma (International Mesothelioma Interest Group stage I), there was no sign of relapse 48 months after treatment (44 mg/m2 body surface area). CONCLUSIONS: The administration of intracavitary cisplatin-fibrin is safe with favorable pharmacokinetics. Although most patients had advanced disease, long-term outcomes are comparable to other multimodal concepts. A confirmation phase II trial is ongoing.

The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8.

The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97-ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97-ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97-ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.

Heterogeneity in Malignant Pleural Mesothelioma.

Despite advances in malignant pleural mesothelioma therapy, life expectancy of affected patients remains short. The limited efficiency of treatment options is mainly caused by inter- and intra-tumor heterogeneity of mesotheliomas. This diversity can be observed at the morphological and molecular levels. Molecular analyses reveal a high heterogeneity (i) between patients; (ii) within different areas of a given tumor in terms of different clonal compositions; and (iii) during treatment over time. The aim of the present review is to highlight this diversity and its therapeutic implications.

Low Merlin expression and high Survivin labeling index are indicators for poor prognosis in patients with malignant pleural mesothelioma.

INTRODUCTION: Alterations of the tumor suppressor Neurofibromatosis type II (NF2) have been reported in about 40% of Malignant pleural mesothelioma (MPM) patients. NF2 (Merlin) deficiency leads to alterations of the Hippo pathway; resulting in activation of the oncogenic Yes Associated Protein-1 (YAP1). Our aim was to investigate the association between these alterations and clinical outcomes. MATERIAL AND METHODS: Tissue microarrays composed of MPM tumors derived from 2 independent MPM cohorts were employed for this study. Immunohistochemical expression of Merlin, YAP1 and its target genes, Survivin and connective tissue growth factor (CTGF) were assessed in nuclear and cytoplasmic fractions. Cohort 1 was comprised of 145 patients intended to be treated with chemotherapy (CTX) followed by extrapleural pneumonectomy (EPP), thus both pre- and post-CTX tissues were available. Cohort 2 was comprised of 59 patients treated with EPP followed by intraoperative hyperthermic cisplatin and/or adjuvant CTX and/or radiotherapy. Marker expression was quantified by means of labeling index (%) for nuclear Survivin and by H-score for the other markers. The dichotomized marker expression was tested for the association with overall survival (OS) and freedom from recurrence (FFR). RESULTS: Kaplan-Meier survival curves revealed a significant association between low cytoplasmic Merlin expression in pre-induction CTX tissues of cohort 1 with shorter FFR (p = 0.02) and OS (p = 0.03). The same tendency was observed in the chemotherapy naïve tissues obtained during EPP of cohort 2. Low nuclear Merlin expression in post-CTX tissues (available from cohort 1 only) was associated with shorter FFR (p = 0.04) and OS (p = 0.05). High nuclear Survivin labeling indices in both pre- and post-CTX tissues of cohort 1 was associated with shorter FFR (p = 0.02). In cohort 2, this was associated with both FFR and OS (p = 0.046 and p = 0.002, respectively). In multivariate analysis, low expression of cytoplasmic Merlin remained an independent prognosticator for shorter FFR of cohort 1 [hazard ratio (HR) = 0.5, 95% confidence interval (CI) = 0.3-0.9, p = 0.001] and OS [HR = 0.5, 95% CI = 0.3-1, p = 0.04]. High Survivin labeling index was an independent prognostic factor for shorter FFR in patients from cohort 1 [HR = 3.4, 95% CI = 1.7-6.8, p = 0.006] and shorter OS in patients from cohort 2 [HR = 2.35, 95% CI = 1.27-4.33, p = 0.006]. CONCLUSIONS: Our findings uncover the significance of Merlin protein expression and Survivin labeling index as prognosticators for poor clinical outcome in two independent MPM cohorts. If confirmed, these markers may be used to identify subgroups of patients benefitting from additional treatment.

Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma.

An autocrine-driven upregulation of the Hedgehog (Hh) signaling pathway has been described in malignant pleural mesothelioma (MPM), in which the ligand, desert Hh (DHH), was produced from tumor cells. However, our investigation revealed that the Hh pathway is activated in both tumor and stroma of MPM tumor specimens and an orthotopic immunocompetent rat MPM model. This was demonstrated by positive immunohistochemical staining of Glioma-associated oncogene 1 (GLI1) and Patched1 (PTCH1) in both tumor and stromal fractions. DHH was predominantly expressed in the tumor fractions. To further investigate the role of the Hh pathway in MPM stroma, we antagonized Hh signaling in the rat model of MPM using a Hh antagonist, vismodegib, (100 mg/kg orally). Daily treatment with vismodegib efficiently downregulated Hh target genes Gli1, Hedgehog Interacting Protein (Hhip), and Ptch1, and caused a significant reduction of tumor volume and tumor growth delay. Immunohistochemical analyses revealed that vismodegib treatment primarily downregulated GLI1 and HHIP in the stromal compartment along with a reduced expression of previously described fibroblast Hh-responsive genes such as Fibronectin (Fn1) and Vegfa Primary cells isolated from the rat model cultured in 3% O2 continued to express Dhh but did not respond to vismodegib in vitro However, culture supernatant from these cells stimulated Gli1, Ptch1, and Fn1 expression in mouse embryonic fibroblasts, which was suppressed by vismodegib. Our study provides new evidence regarding the role of Hh signaling in MPM stroma in the maintenance of tumor growth, emphasizing Hh signaling as a treatment target for MPM. Mol Cancer Ther; 15(5); 1095-105. ©2016 AACR.

Hedgehog Signaling in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a cancer associated with exposure to asbestos fibers, which accumulate in the pleural space, damage tissue and stimulate regeneration. Hedgehog signaling is a pathway important during embryonic mesothelium development and is inactivated in adult mesothelium. The pathway is reactivated in some MPM patients with poor clinical outcome, mainly mediated by the expression of the ligands. Nevertheless, mutations in components of the pathway have been observed in a few cases. Data from different MPM animal models and primary culture suggest that both autocrine and paracrine Hedgehog signaling are important to maintain tumor growth. Drugs inhibiting the pathway at the level of the smoothened receptor (Smo) or glioma-associated protein transcription factors (Gli) have been used mostly in experimental models. For clinical development, biomarkers are necessary for the selection of patients who can benefit from Hedgehog signaling inhibition.

Importance of excision repair cross-complementation group 1 and ribonucleotide reductase M1 as prognostic biomarkers in malignant pleural mesothelioma treated with platinum-based induction chemotherapy followed by surgery.

OBJECTIVES: Survival and response to platinum-based induction chemotherapy are heterogeneous among patients with malignant pleural mesothelioma. The aim of the present study was to assess the prognostic role of DNA repair markers, such as excision repair cross-complementation group 1 and ribonucleotide reductase M1, in multimodally treated patients with malignant pleural mesothelioma. METHODS: Tumor tissue of a malignant pleural mesothelioma cohort (n = 107) treated with platinum/gemcitabine (n = 46) or platinum/pemetrexed (n = 61) induction chemotherapy followed by extrapleural pneumonectomy was assembled on a tissue microarray. Immunohistochemical expression of excision repair cross-complementation group 1 (nuclear) and ribonucleotide reductase M1 (nuclear and cytoplasmic) was assessed for its prognostic impact (association with overall survival or freedom from recurrence). RESULTS: Patients with high nuclear ribonucleotide reductase M1 expression before chemotherapy showed significantly longer freedom from recurrence (P = .03). When specifically analyzed in the subgroup of patients receiving platinum/gemcitabine followed by extrapleural pneumonectomy, high nuclear ribonucleotide reductase M1 was associated with prolonged freedom from recurrence (P = .03) and overall survival (P = .02). Low excision repair cross-complementation group 1 expression in prechemotherapy tumor tissues was associated with significantly longer freedom from recurrence (P = .04). Nuclear ribonucleotide reductase M1 and excision repair cross-complementation group 1 were independent prognosticators of freedom from recurrence in addition to pT stage in multivariate analysis. CONCLUSIONS: In the present study, nuclear ribonucleotide reductase M1 and excision repair cross-complementation group 1 expression were identified as independent prognosticators for freedom from recurrence of malignant pleural mesothelioma in patients undergoing induction chemotherapy followed by extrapleural pneumonectomy.

Evaluation of imaging techniques for the assessment of tumour progression in an orthotopic rat model of malignant pleural mesothelioma†.

OBJECTIVES: An orthotopic rat tumour recurrence model for malignant pleural mesothelioma (MPM) provides clinical similarity to patients and is useful for drug testing combined with surgical intervention. Importantly, a reliable imaging method is required allowing for noninvasive and repetitive evaluation of the tumour load. We compared the tumour load assessed by bioluminescence and magnetic resonance imaging (MRI) to the macroscopic tumour volume as a reference standard. METHODS: A total of 500,000 syngeneic rat MPM cells transfected with luciferase were implanted underneath the parietal pleura of immunocompetent rats (n=13). From the second day after implantation, bioluminescence measurements of the tumour load expressed as the maximum bioluminescent intensity (photon/second) were performed daily after intraperitoneal injection of the luciferase substrate, d-luciferin, to observe the first occurrence of tumour. Six days after the first detection of tumour, bioluminescence, MRI and macroscopic tumour volume measurement were conducted. For MRI, a 4.7-Tesla small animal imager equipped with a 1H whole-body rat coil was employed using T2-weighted fast spin-echo sequences. Tumour burden (mm3) was quantified from magnetic resonance transverse images by two independent readers by manual segmentation. Finally, the tumour burden assessed by bioluminescence and MRI was correlated (Pearson's correlation) with the macroscopic measurement of tumour (ellipsoid) volume. RESULTS: In all rats, a single tumour nodule was found at the inoculation site with a median macroscopic volume of 46 mm3 (18-377 mm3). For tumour burden quantification of MRIs, we observed good interobserver correlation (R2=0.81, P<0.0001) as well as significant association with the macroscopic tumour volume (R2=0.59, P=0.002). However, the signal intensity of bioluminescence did not correspond to the macroscopic tumour volume (R2=0.01, P=0.76). CONCLUSIONS: MRI is a reliable and reproducible noninvasive in vivo imaging method for MPM tumour burden assessment for the present MPM model.

PI3K/mTOR signaling in mesothelioma patients treated with induction chemotherapy followed by extrapleural pneumonectomy.

INTRODUCTION: The prognostic significance of activity biomarkers within the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway was assessed in two independent cohorts of malignant pleural mesothelioma (MPM) patients uniformly treated with a multimodal approach. We specifically assessed expression signatures in a unique set of pre- and postchemotherapy tumor samples. METHODS: Biomarker expression was assessed in samples of two independent cohorts of 107 (cohort 1) and 46 (cohort 2) MPM cases uniformly treated with platinum-based induction chemotherapy followed by extrapleural pneumonectomy from two different institutions, assembled on tissue microarrays. Expression levels of phosphatase and tensin homologue (PTEN), phospho-mTOR, and p-S6 in addition to marker of proliferation (Ki-67) and apoptosis (cleaved caspase-3) were evaluated by immunohistochemistry and correlated with overall survival (OAS) and progression-free survival (PFS). To assess PTEN genomic status, fluorescence in situ hybridization was performed. RESULTS: Survival analysis showed that high p-S6 and Ki-67 expression in samples of treatment naïve patients of cohort 1 was associated with shorter PFS (p = 0.02 and p = 0.04, respectively). High Ki-67 expression after chemotherapy remained associated with shorter PFS (p = 0.03) and OAS (p = 0.02). Paired comparison of marker expression in samples before and after induction chemotherapy of cohort 1 revealed that decreased cytoplasmic PTEN and increased phospho-mTOR expression was associated with a worse OAS (p = 0.04 and p = 0.03, respectively). CONCLUSIONS: These novel data reveal a prognostic significance of expression changes of PI3K/mTOR pathway components during induction chemotherapy if confirmed in other patient cohorts and support the growing evidence to target the PI3K/mTOR pathway in the treatment of MPM.

The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks.

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion. Protein modification with ubiquitin is crucial for the signalling cascade, but how ubiquitylation coordinates the dynamic assembly of these complexes is poorly understood. Here, we show that the human ubiquitin-selective protein segregase p97 (also known as VCP; valosin-containing protein) cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signalling complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1-NPL4 and Lys-48-linked ubiquitin (K48-Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48-Ub conjugates from sites of DNA damage to orchestrate proper association of 53BP1, BRCA1 and RAD51, three factors critical for DNA repair and genome surveillance mechanisms. Impairment of p97 activity decreases the level of DSB repair and cell survival after exposure to ionizing radiation. These findings identify the p97-UFD1-NPL4 complex as an essential factor in ubiquitin-governed DNA-damage response, highlighting its importance in guarding genome stability.

Degradation-linked ubiquitin signal and proteasome are integral components of DNA double strand break repair: New perspectives for anti-cancer therapy.

Damaged DNA leads to genomic instability that causes many diseases such as cancer. Cells evolved the DNA damage response (DDR), which recognizes and efficiently repairs damaged DNA through the action of highly coordinated signalling mechanisms. Recently, a non-degradation-linked Lys(K)63-ubiquitin signal emerged as a signalling pathway essential for orchestration of the DDR after DNA double strand breaks (DSBs). How the ubiquitin-dependent proteasomal degradation system (UPS) coordinates DDR after DSBs is still poorly understood. Here, we review the evidence, suggesting the involvement of the degradation-linked K48-ubiquitin signal and the proteasome at the sites of DSBs. Based on this we propose the UPS as a central element in the orchestration of the DDR at the sites of DSBs. The suggested model is also discussed in the context of anti-cancer therapy.

Accumulation of lipid peroxidation-derived DNA lesions in iron-overloaded thalassemic mouse livers: comparison with levels in the lymphocytes of thalassemia patients.

In thalassemia patients, iron overload can stimulate lipid peroxidation (LPO), thereby generating miscoding DNA adducts. Adducted DNA was measured in the lymphocytes of beta-Thal/Hb E patients and healthy controls and in the organs of thalassemic mice. epsilondA, epsilondC and M(1)dG residues were quantified by (32)P-postlabeling-TLC/HPLC. M(1)dG levels in lymphocyte DNA from patients were 4 times as high as in controls, while the increase in epsilondA and epsilondC was not significant. Adducted DNA accumulated in the liver of thalassemic mice having >2.7 mg Fe/g tissue dry weight; DNA adducts and iron were highly correlated. epsilondA was not specifically generated in certain mouse liver cell types as revealed by immunohistochemical staining. We found elevated LPO-induced DNA damage in the liver of thalassemic mouse and in lymphocytes, implicating that massive DNA damage occurs in the liver of thalassemia patients. We conclude that promutagenic LPO-derived DNA lesions are involved in the onset of hepatocellular carcinoma in these patients.

Increased urinary 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine excretion in thalassemia patients: markers for lipid peroxidation-induced DNA damage.

Thalassemic diseases including homozygous beta-thalassemia and beta-thalassemia/Hb E (beta-Thal/Hb E) are prevalent in Southeast Asia. Iron overload is a common complication in beta-thalassemia patients which induces intracellular oxidative stress and lipid peroxidation (LPO). LPO end products generate miscoding etheno adducts in DNA which after their repair are excreted in urine. We investigated whether urinary levels of 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) can serve as putative cancer risk markers in beta-Thal/Hb E patients. epsilondA and epsilondC levels were assayed in collected urine samples by immunoprecipitation-HPLC-fluorescence and 32P-postlabeling TLC, respectively. Mean epsilondA (fmol/micromol creatinine) levels in urine of beta-Thal/Hb E patients ranged from 4.8 to 120.4 (33.8+/-3.9; n=37) and were 8.7 times higher compared to asymptomatic controls (1.4-13.8; 3.9+/-0.8; n=20). The respective epsilondC levels ranged from 0.15 to 32.5 (5.2+/-1.3; n=37) and were increased some 13 times over controls (0.04-1.2; 0.4+/-0.7; n=20). epsilondC levels were correlated positively with NTBI (r=0.517; P=0.002), whereas epsilondA showed only a trend (r=0.257; P=0.124). We conclude that the strongly increased urinary excretion of etheno adducts indicates elevated LPO-induced DNA damage in internal organs such as the liver. These highly promutagenic lesions may contribute to the increased risk of thalassemia patients to develop hepatocellular carcinoma.