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Aim: Clinical features of esophageal cancer (EC) patients have poor prognostic power. Thus, it is paramount to discover biomarkers that can allow a more accurate survival prediction. Methods: To detect genetic variants associated with survival, DNA from 120 patients treated with cisplatin-based neoadjuvant therapy were genotyped using drug metabolism enzymes and transporters array. Results: We identified two variants: the rs2038067 in PPARD (p = 0.0004) and the rs683369 (F160L) in SLC22A1 (p = 0.001). Their prognostic power was greater than that of clinical stage alone (p = 0.017) and comparable to that of response to neoadjuvant therapy (p = 0.71). Interestingly, the prognostic accuracy of response models increased significantly when genetic variables were included (p = 0.003). Conclusion: Our data, though preliminary, strengthen the potential utility of germline variants for a better-tailored management of EC patients.

Liquid biopsy is currently approved for management of epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) patients. However, one unanswered question is whether the rate of cell-free DNA (cfDNA)-negative samples is due to technical limitations rather than to tumor genetic characteristics. Using four microsatellite markers that map specific chromosomal loci often lost in lung cancer, we conducted a pilot study to investigate whether other alterations, such as loss of heterozygosity (LOH), could be detected in EGFR-negative cfDNA. We analyzed EGFR-mutated NSCLC patients (n = 24) who were positive or negative for EGFR mutations in cfDNA and compared the results with a second cohort of 24 patients bearing KRAS-mutated cancer, which served as a representative control population not exposed to targeted therapy. The results showed that in EGFR-negative post-tyrosine-kinase-inhibitor (TKI) cfDNAs, LOH frequency was significantly higher than in both pre- and post-TKI EGFR-positive cfDNAs. By contrast, no association between KRAS status in cfDNA and number of LOH events was found. In conclusion, our study indicates the feasibility of detecting LOH events in cfDNA from advanced NSCLC and suggests LOH analysis as a new candidate molecular assay to integrate mutation-specific assays.

Esophageal cancer (EC) is a very aggressive tumor, and no reliable prognostic markers exist especially for resectable advanced neoplasia. The principal aim of this study was to investigate the association of germline polymorphisms in nucleotide excision repair (NER) pathway genes with the overall survival (OS) of patients with advanced EC. As a second aim, we also studied the association of NER gene variants with response to cisplatin-based chemotherapy. Among the EC patients referred to our Institution between 2004 and 2012, we selected a cohort of 180 patients diagnosed with a clinical tumor stage ranging from IIB and IVA. Patients were genotyped for four NER variants, two in the ERCC1 (rs11615 and rs3212986) and two in the ERCC2/XPD (rs1799793 and rs13181) genes. Kaplan–Meier analyses and Cox proportional hazards model were used to evaluate the associations of the selected variants with OS; association with response to neoadjuvant therapy was investigated using logistic regression. Results showed that the ERCC1 rs3212986 and the ERCC2/XPD rs1799793 were significantly associated with shorter OS. On the contrary, response association analysis displayed that, while rs11615 and rs3212986 in ERCC1 were associated with response, both ERCC2/XPD variants were not. By creating survival prediction models, we showed that the rs3212986 and the rs1799793 have a better predictability of the tumor stage alone. Furthermore, they were able to improve the power of the clinical model (AUC = 0.660 vs. AUC = 0.548, p = 0.004). In conclusion, our results indicate that the ERCC1 rs3212986 and the ERCC2/XPD rs1799793 could be used as surrogate markers for a better stratification of EC patients with advanced resectable tumor.

So far, no reliable predictive clinicopathological markers of response to aromatase inhibitors (AIs) have been identified, and little is known regarding the role played by host genetics. To identify constitutive predictive markers, an array-based association study was performed in a cohort of 55 elderly hormone-dependent breast cancer (BC) patients treated with third-generation AIs. The array used in this study interrogates variants in 225 drug metabolism and disposition genes with documented functional significance. Six variants emerged as associated with response to AIs: three located in ABCG1, UGT2A1, SLCO3A1 with a good response, two in SLCO3A1 and one in ABCC4 with a poor response. Variants in the AI target CYP19A1 resulted associated with a favourable response only as haplotype; haplotypes with increased response association were also detected for ABCG1 and SLCO3A1. These results highlight the relevance of host genetics in the response to AIs and represent a first step toward precision medicine for elderly BC patients.

Barrett's esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma. Despite the low absolute risk of neoplastic progression of BE, probability increases with the diagnosis of dysplasia. For this reason, BE patients undergo an endoscopy-based surveillance that is, however, burdensome for patients, subject to inter-observer subjectivity, and expensive for national health systems. Thus, less invasive and low-cost diagnostic tools are needed. This study is aimed at finding a simple and reliable method to detect in the circulating cell-free DNA (cfDNA) of BE patients evidence of the molecular instability that accompanies BE carcinogenesis. We chose the loss of heterozygosity analysis because chromosomal region gains or losses have been described in BE and esophageal adenocarcinoma. Furthermore, this analysis does not require an a priori knowledge of tumor specific mutations and/or rearrangements. Previous data showed a good consistency between tissue and cfDNA alterations. Here, we report that, in the cfDNA of dysplastic BE patients, the frequency of genetic alterations is statistically higher than that of metaplastic BE patients (P = 0.005). Interestingly, after endoscopic treatment, the alteration frequency dropped, suggesting that cfDNA can also be used to monitor curative effects. Among the used markers, those that map nearby TP53 gene were the most discriminant between metaplastic and dysplastic BE. Furthermore, longitudinal follow-up cases showed that genetic alterations can be found in cfDNA before the appearance of a detectable lesion. Altogether, our data suggest that the use of liquid biopsy could become a minimally invasive diagnostic tool to implement BE patient monitoring.

Chemoradiotherapy followed by surgery is at present the standard therapeutic approach for esophageal cancer (EC) in patients with resectable tumor. However, response to neoadjuvant therapy is characterized by a strong interpatient variability, and the identification of markers predictive of outcome is mandatory. In this review, taking into account the currently available literature, we report the impact that host genetic variables can have on EC neoadjuvant therapy. We mainly focused on the gene variants involved in the pharmacokinetics and pharmacodynamics of the common chemotherapeutic drugs used to treat EC patients, commented on the weakness of the present knowledge, and discussed the future strategies to achieve a more personalized and effective EC treatment.

Barrett's esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma. For this reason, endoscopic-based surveillance protocols have been developed. This prevention program is, however, burdensome for the patients and expensive for the national health systems. Thus, diagnostic strategies with a low invasiveness and a reduced economic impact are required. This study investigated the power of plasma circulating free DNA (cfDNA) in predicting neoplastic transformation in the natural history of two BE patients who progressed to esophageal adenocarcinoma. Longitudinally collected DNAs from plasma and paired formalin fixed paraffin embedded samples were examined for both loss of heterozygosity (LOH) in areas proximal to TP53, FHIT and BRCA2 genes, and mutations in TP53 gene. Results showed that: (i) early BE molecular alterations are mainly localized proximal to, or within, TP53 gene; (ii) LOH events present in cfDNA not only retrace the time-matched biopsy profile but better represent the total alterations of the BE epithelium. In conclusion, our findings suggested that LOH analysis in plasma cfDNA could represent an additional, less invasive, diagnostic tool to monitor neoplastic progression of BE epithelium.

The occurrence of a second primary esophageal carcinoma (EC) in long-term cancer survivors may represent a late effect of previous radio-chemotherapeutic treatment. To identify the genetic factors that could increase this risk, we analyzed nine variants within ERCC1, XPD, XRCC1 and XRCC3 DNA repair pathway genes, and GSTP1, TP53 and MDM2 genes in 61 patients who received radio-chemotherapy for a prior lymphoma or breast cancer; 29 of them had a second primary EC. This cohort consists of 22 esophageal squamous cell carcinoma (ESCC) and 7 esophageal adenocarcinoma (EADC) patients. A validation cohort of 154 patients with sporadic EC was also included. The XPD Asp312Asn (rs1799793) was found to be associated with the risk of developing second primary ESCC (P=0.015). The resultant variant was also involved in the onset of sporadic ESCC (P=0.0018). To know in advance who among long-term cancer survivors have an increased risk of EC could lead to a more appropriate follow-up strategy.

Background Temozolomide (TMZ) administered daily with radiation therapy (RT) for 6 weeks, followed by adjuvant TMZ for 6 cycles, is the standard therapy for newly diagnosed glioblastoma (GBM) patients. Although TMZ is considered to be a safe drug, it has been demonstrated to cause severe myelotoxicity; in particular, some case reports and small series studies have reported severe myelotoxicity developing during TMZ and concomitant RT. We performed a prospective study to analyze the incidence of early severe myelotoxicity and its possible clinical and genetic factors. Patients and methods From November 2010 to July 2012, newly diagnosed GBM patients were enrolled. They were eligible for the study if they met the following criteria: pathologically proven GBM, age 18 years and older, an Eastern Cooperative Oncology Group performance status of 0 to 2, adequate renal and hepatic function, and adequate blood cell counts before starting TMZ plus RT. Grading of hematologic toxicity developing during radiation and TMZ was based on the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. Clinical factors from all patients were recorded. The methylation status and polymorphic variants of O-methylguanine-DNAmethyl-transferase gene in peripheral blood mononuclear cells, and polymorphic genetic variants of genes involved in the pharmacokinetics and pharmacodynamics of TMZ, were analyzed. For genetic analyses, patients with toxicity were matched (1:2) for age, performance status, anticonvulsants, and proton pump inhibitors with patients without myelotoxicity. Results We enrolled 87 consecutive GBM patients: 32 women and 55 men; the average age was 60 years. During TMZ and RT, 4 patients (5%) showed grade 3-4 myelotoxicity, and its median duration was 255 days. Predictor factors of severe myelotoxicity were female sex, pretreatment platelet count of ≤3,00,000/mm, methylated O-methylguanine-DNA methyltransferase promoter in the hematopoietic cell system, and specific polymorphic variants of the cytochrome P450 oxidoreductase and methionine adenosyltransferase 1A genes. Conclusions Although we studied a small population, we suggest that both clinical and genetic factors might simultaneously be associated with severe myelosuppression developed during TMZ plus RT. However, our results deserve validation in larger prospective studies and, if the factors associated with severe myelotoxicity are validated, dose adjustments of TMZ for those patients may reduce the risk of severe myelotoxicity during the concomitant treatment.

Defects in the regulation of programmed cell death play a fundamental role in the development of neoplasia and neurological disorders, both of which are linked to the human T-cell leukemia/lymphoma virus type 1 (HTLV-1) infection. We previously showed that the HTLV-1 Tax protein protects from apoptosis induced by serum starvation by preventing cytochrome c release and Bax relocation to mitochondria, two early events in the mitochondrial apoptotic pathway. As a natural extension of these findings, and to better define the action of Tax, in the present study, we investigated the outcome of Tax and two mutants which are inactive in CREB/ATF (M47) or NF-κB (M22) pathways, in the control of apoptosis induced by the proapoptotic Bax protein. We found that activation of CREB, rather than NF-κB, is a key phenomenon in preventing apoptosis. Furthermore, the importance of CREB activation is strengthened by experiments with CREB mutants, treatment with forskolin, and in situ analysis of P-CREB status in cells transfected with Tax or its nonprotecting M47 mutant. Considered together, these results underscore a primary role of CREB in preventing apoptosis triggered by Bax, and suggest that Tax might act by affecting the phosphorylation state of CREB.

Deregulation of the apoptotic process can lead to pathophysiological changes that result in either degenerative diseases or cancer. Although the transactivator Tax has been established as an essential effector of human T-lymphotropic virus type-1 (HTLV-1)-mediated diseases, which include both a neurodegenerative pathology and leukemia/lymphoma, its exact role(s) in the pathogenesis remains to be clarified. Because the apoptotic potential of Tax is still being debated, we addressed this question by testing the susceptibility of Tax(-) and Tax(+) cells to apoptosis under conditions of growth factor withdrawal and Bax overexpression. Results showed that Tax(+) cells are protected from apoptosis triggered by depletion of growth factors. This protective effect seems to be the result of a block in the apoptotic program regulated by mitochondria. Furthermore, in an attempt to elucidate which transcriptional pathway activated by Tax is important in the observed Tax-induced resistance, we found that CREB/ATF activity plays a relevant role in protecting cells from apoptosis induced by Bax overexpression. All together, these data might suggest that the ability of Tax to inhibit certain apoptotic stimuli could be important in its role as a viral transforming protein.

Transgenic mice carrying the tax gene of human T-cell lymphotropic virus type I displayed a high prevalence of arthropathy. The percentage of affected animals increased with age, reaching a peak of 43% at 20 months. Southern analysis of deoxyribonucleic acid (DNA) from tissue samples indicated that disease development was related to tax copy number. Histopathologic evaluation of the ankle joints disclosed deep erosion of the synovial lining, fibrous tissue proliferation together with angiogenesis, mononuclear cell infiltration, and activation of osteoclasts. Radiologic examination confirmed joint involvement and revealed bone architecture modifications. The phenotype exhibited by the affected animals closely resembles that of seronegative arthritis in humans, and may indicate tax protein as a causal agent of arthropathy observed in HTLV-I infected individuals.

At present, no consensus exists on the beneficial effect of preoperative cisplatin/5-fluorouracil (5-FU)-based chemotherapy versus primary surgery in the management of patients with esophageal cancer. The aim of this study was to evaluate the impact of some relevant genetic polymorphisms, within drug-related and DNA repair genes, on the clinical outcome of esophageal cancer patients subjected to cisplatin/5-FU-based neoadjuvant treatment.DNA from 143 esophageal cancer patients, 63 receiving neoadjuvant therapy and 80 receiving primary surgery, was analyzed for the following polymorphisms: the GSTM1 null, GSTT1 null, and GSTP1 Ile105Val (rs16953) in glutathione S-transferase (GST) family, 2 in thymidylate synthase (TS) gene, and the ERCC1 Asn118Asn (rs11615), ERCC1 C8092A (rs3212986), XPD/ERCC2 Asp312Asn (rs1799793), and XPD/ERCC2 Lys751Gln (rs13181) of the nucleotide excision repair pathway.We found that the ERCC1 rs3212986, although not associated with therapeutic response, is an independent predictive marker of better outcome in a cisplatin/5-FU-based neoadjuvant setting (hazard ratio: 0.38, 95% confidence interval: 0.2-0.73, P=0.008). In contrast, no association with clinical outcome was observed for this polymorphism in the primary surgery group.Our study indicates the ERCC1 rs3212986 as a predictive marker in the cisplatin/5-FU-based neoadjuvant setting, and also suggests its use as a marker to select the appropriate therapeutic approach in esophageal cancer patients.

5-fluorouracil (5-FU) has been widely used since the 1980s, and it remains the backbone of many chemotherapeutic combination regimens. However, its use is often limited by the occurrence of severe toxicity. Although several reports have shown the detrimental effect of some dihydropyrimidine dehydrogenase (DPYD) and thymidylate synthase (TYMS) gene polymorphisms in patients undergoing 5-FU-based treatment, they account for only a minority of toxicities. Looking for new candidate genetic variants associated with 5-FU-induced toxicity, we used the innovative genotyping microarray Affymetrix Drug-Metabolizing Enzymes and Transporters (DMET)™ Plus GeneChip that interrogates 1,936 genetic variants distributed in 231 genes involved in drug metabolism, excretion, and transport. To reduce variability, we analyzed samples from colorectal cancer patients who underwent fairly homogenous treatments (i.e., Machover or Folfox) and experienced G3 or G4 toxicity; control patients were matched for therapy and selected from those who did not disclose toxicity (G0–G1). Pharmacogenetic genotyping showed no significant difference in DPYD and TYMS genetic variants distribution between cases and controls. However, other polymorphisms could account for 5-FU-induced toxicity, with the CHST1 rs9787901 and GSTM3 rs1799735 having the strongest association. Although exploratory, this study suggests that genetic polymorphisms not directly related to 5-FU pharmacokinetics and pharmacodynamics are involved in 5-FU-induced toxicity. Our data also indicates DMET™ microarray as a valid approach to discover new genetic determinants influencing chemotherapy-induced toxicity.

Platinum-based neoadjuvant therapy is the standard treatment for esophageal cancer (EC). At present, no reliable response markers exist, and patient therapeutic outcome is variable and very often unpredictable. The aim of this study was to understand the contribution of host constitutive DNA polymorphisms in discriminating between responder and nonresponder patients. DNA collected from 120 EC patients treated with platinum-based neoadjuvant chemotherapy was analyzed using drug metabolism enzymes and transporters (DMET) array platform that interrogates polymorphisms in 225 genes of drug metabolism and disposition. Four gene variants of DNA repair machinery, 2 in ERCC1 (rs11615; rs3212986), and 2 in XPD (rs1799793; rs13181) were also studied. Association analysis was performed with pTest software and corrected by permutation test. Predictive models of response were created using the receiver-operating characteristics curve approach and adjusted by the bootstrap procedure. Sixteen single nucleotide polymorphisms (SNPs) of the DMET array resulted significantly associated with either good or poor response; no association was found for the 4 variants mapping in DNA repair genes. The predictive power of 5 DMET SNPs mapping in ABCC2 , ABCC3 , CYP2A6 , PPARG , and SLC7A8 genes was greater than that of clinical factors alone (area under the curve [AUC] = 0.74 vs 0.62). Interestingly, their combination with the clinical variables significantly increased the predictivity of the model (AUC = 0.78 vs 0.62, P = 0.0016). In conclusion, we identified a genetic signature of response to platinum-based neoadjuvant chemotherapy in EC patients. Our results also disclose the potential benefit of combining genetic and clinical variables for personalized EC management.

Ras signaling pathways play an important role in cellular proliferation and survival, and inappropriate activation of Ras frequently results in cell transformation and cancer. Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is the etiological agent of the adult T-cell leukemia/lymphoma (ATLL), a severe malignancy that has a poor prognosis and exhibits resistance to conventional chemotherapy. Although the mechanisms involved in cell transformation by HTLV-1 have not been completely clarified, it is generally thought that Tax plays a pivotal role in the process. We have previously proposed that a functionally active Ras protein is needed for efficient anti-apoptotic activity of Tax. In this study we report data indicating that the apoptotic resistance of cells expressing Tax, constitutively or transiently, is linked to the intracellular levels of Ras-GTP. Indeed, we found that Tax-positive cells have a high content of active Ras, and that inhibition of Ras signaling, using the antagonist farnesyl thyosalicylic acid (FTS), increases their sensitivity to apoptosis. FTS treatment was also accompanied by a decrease in ERK, but not Akt, phosphorylation. Thus, all together our data suggest that the interaction between Tax and Ras could be important to ATLL pathogenesis, and indicate Ras as a possible target for therapeutic intervention in ATLL patients.

We report multiplex ligation-dependent probe amplification analysis (MLPA) of DNA copy number alterations in 59 esophageal cancer samples, stratified by histotype. Results showed that squamous cell carcinoma (SCC) samples present clustered abnormalities with several genes altered at high frequency. Instead, esophageal adenocarcinoma (ADC) samples are characterized by a more widespread genomic instability, and in these patients total DNA copy number alterations resulted to be an independent prognostic factor. The detection of characteristic molecular changes represents a step towards a better understanding of the molecular basis of esophageal tumorigenesis, and might offer the potential for the discovery of tumor-specific biomarkers.

Analyzing the mechanisms underlying the capability of the monosialoganglioside GM1 to induce CD4 modulation we observed that GM1 has a dual effect on the CD4 molecule. GM1 treatment of the lymphoma cell line MOLT-3 and CD4-transfected HeLa cells for times shorter than 30 min prevented binding of monoclonal antibodies (mAbs) recognizing epitopes located within the first NH2-terminal domains of CD4, but not of the OKT4 mAb, which binds to the region of CD4 proximal to the transmembrane domain. However, no binding of the OKT4 mAb was observed after a few hours of treatment with GM1 in both MOLT-3 cells and HeLa cells transfected with an intact CD4 molecule, but not in HeLa cells transfected with a CD4 molecule lacking the bulk of the cytoplasmic domain, suggesting that modulation of CD4 by GM1 depends on the integrity of the cytoplasmic domain. GM1 treatment blocked binding of several mAbs which recognize epitopes located within the first two NH2-terminal domains of CD4 and did not induce CD4 down-modulation if MOLT-3 cells were preincubated with the OKT4A or the OKT4 mAbs. Immunoprecipitation studies with [35S]methionine-labeled MOLT-3 cells showed that GM1-induced CD4 down-modulation was accompanied by CD4 degradation, and this was preceded by dissociation of p56lck from CD4. GM1-induced CD4 down-modulation, dissociation of p56lck from CD4, and CD4 degradation were unaffected by staurosporine, which, on the contrary, blocked these events in response to phorbol 12-myristate 13-acetate. These observations demonstrate that the first action of GM1 is to mask epitopes located within the first two NH2-terminal domains; then, GM1 triggers protein kinase C-independent signals which cause p56lck dissociation from CD4 and the delivery of the molecule to an intracellular compartment where it is eventually degraded.

The present study investigated the function of p13, a mitochondrial protein of human T-cell leukemia virus type 1 (HTLV-1). Although necessary for viral propagation in vivo, the mechanism of function of p13 is incompletely understood. Drawing from studies in isolated mitochondria, we analyzed the effects of p13 on mitochondrial reactive oxygen species (ROS) in transformed and primary T cells. In transformed cells (Jurkat, HeLa), p13 did not affect ROS unless the cells were subjected to glucose deprivation, which led to a p13-dependent increase in ROS and cell death. Using RNA interference we confirmed that expression of p13 also influences glucose starvation-induced cell death in the context of HTLV-1–infected cells. ROS measurements showed an increasing gradient from resting to mitogen-activated primary T cells to transformed T cells (Jurkat). Expression of p13 in primary T cells resulted in their activation, an effect that was abrogated by ROS scavengers. These findings suggest that p13 may have a distinct impact on cell turnover depending on the inherent ROS levels; in the context of the HTLV-1 propagation strategy, p13 could increase the pool of “normal” infected cells while culling cells acquiring a transformed phenotype, thus favoring lifelong persistence of the virus in the host.

We describe two cases of adult T-cell leukemia (ATL) in young adult males. One patient was born in the Seychelles Islands and came to live in Italy at 15 years of age; five years later, he started to experience several episodes of infestation by Strongyloides stercoralis, and opportunistic infections; at the age of 25, enlarged lymph nodes were first documented, and he died one month later, with a final diagnosis of ATL. The other patient was born in Rumania. At the age of 34 years, he presented with asthenia, liver and lymph node enlargement, lymphocytosis and cutaneous lesions, and a diagnosis of malignant lymphoma was made; despite chemotherapy, he died five months later. Serologic analyses demonstrated the presence of anti-HTLV-I antibodies in both patients, but not anti-HIV-1 antibodies. HTLV-1 nucleotide sequences were detected in the neoplastic cells of both patients.

Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) plays a critical role in HTLV-I-correlated diseases through its ability to deregulate the expression of a vast array of cellular genes. We have previously shown that Tax counteracts apoptosis induced by stimuli triggering mitochondria apoptotic pathway, most likely by activating CREB-mediated transcription and affecting the phosphorylation levels of CREB at Ser-133. Here, we report data that indicate the oncoprotein Ras as a possible mediator of Tax-induced apoptosis protection and suggest a possible role of Tax in Ras activation. In addition, using inhibitors of down stream effectors of Ras, we found that ERK signaling is the most relevant for Tax-mediated apoptosis protection. As a whole, our findings provide intriguing evidence of a possible link between Ras signaling and Tax capability to counteract apoptosis and to enhance P-CREB levels, and implicates a potential role for Ras in HTLV-1-induced diseases.

Despite recent advances in surgical and multidisciplinary treatment, prognosis for patients with esophageal adenocarcinoma remains poor, and the low prognostic significance of pTNM staging suggests that additional parameters are needed. To identify genomic abnormalities characteristic of esophageal adenocarcinoma, a panel of 33 samples obtained at surgery from previously untreated patients were analyzed by muliplex ligation-dependent probe amplification technique. We detected frequent gains of 6p, 8q, 13q, 17q, 20q, and losses of 4q, 5q, 15q, and 18q. When DNA copy number changes were correlated to clinicopathological features of patients no association was found between the number of chromosomal aberrations and gender, age, tumor grade or pTNM staging. However, interestingly, a significant correlation between patient survival and total number of chromosomal aberrations was found when esophageal adenocarcinoma cases were stratified according to the median of survival (20 months) (P=0.002) or the median of aberrations (12 aberrations) (P=0.014). Evaluation of the distribution of gains and losses at the level of single chromosomes indicated that gains on chromosomes 5, 6, 8, 11, 20 and losses on chromosomes 1, 3, 5, 11, and 18 were significantly different in the two survival groups. Furthermore, when single gene imbalances were analyzed in further details, we found that besides alterations that involve genes shared by both survival groups, a few genes (KIAA0170, EMS1, ABCC4, F3, and MIF) were altered only in samples from patients with poor survival. Thus, we established a good correlation between the total number of chromosomal alterations and survival, suggesting that the estimation of total imbalances might represent an additional indicator of disease outcome. In addition, the finding of alterations specific for the more aggressive esophageal adenocarcinoma subset might represent promising biomarkers to increase the accuracy of clinical outcome prediction.

Human T cell leukemia virus type 1 encodes an “accessory” protein named p13 II that is targeted to mitochondria and triggers a rapid flux of K + and Ca 2+ across the inner membrane. In this study, we investigated the effects of p13 II on tumorigenicity in vivo and on cell growth in vitro . Results showed that p13 II significantly reduced the incidence and growth rate of tumors arising from c-myc and Ha-ras-cotransfected rat embryo fibroblasts. Consistent with these findings, HeLa-derived cell lines stably expressing p13 II exhibited markedly reduced tumorigenicity, as well as reduced proliferation at high density in vitro . Mixed culture assays revealed that the phenotype of the p13 II cell lines was dominant over that of control lines and was mediated by a heat-labile soluble factor. The p13 II cell lines exhibited an enhanced response to Ca 2+ -mediated stimuli, as measured by increased sensitivity to C2-ceramide-induced apoptosis and by cAMP-responsive element-binding protein (CREB) phosphorylation in response to histamine. p13 II -expressing Jurkat T cells also exhibited reduced proliferation, suggesting that the protein might exert similar effects in T cells, the primary target of HTLV-1 infection. These findings provide clues into the function of p13 II as a negative regulator of cell growth and underscore a link between mitochondria, Ca 2+ signaling, and tumorigenicity.

The human T-cell leukemia virus type 1 (HTLV-1) Tax transactivator is thought to play a primary role in the development of HTLV-1-mediated diseases. Using a murine fibroblast model, we previously showed that Tax reduces apoptosis induced by serum starvation by preventing cytochrome c release from the mitochondria. As Tax can enhance the transcriptional activity of nuclear factor NF-kB and cAMP-responsive element binding protein/activating transcription factor-1 (CREB/ATF-1), we investigated the relevance of these routes in the anti-apoptotic effects of Tax. Results showed that a Tax mutant retaining CREB/ATF-1 transactivating activity protects murine fibroblasts from serum-depletion-induced apoptosis, while two CREB/ATF-1-defective mutants did not. Treatment with forskolin, an activator of CREB, significantly attenuated cytochrome c release and Bax translocation in response of serum deprivation. In analogy to forskolin treatment, Tax expression results in sustained phosphorylation of CREB at Ser(133) during serum starvation. Considered together, these results underscore a primary role of CREB transcriptional activation in preventing apoptosis triggered by growth factor withdrawal, and suggest that Tax might in part function by affecting the phosphorylation state of CREB.

Although the viral transactivator Tax has been established as an essential effector of HTLV-I-mediated oncogenesis, its exact role(s) in the pathogenesis of HTLV-I-associated diseases, which include both a neurodegenerative pathology and leukemia/lymphoma, remains to be clarified. It was recently advanced that dysregulation of the apoptotic process can lead to pathophysiological changes which result in either degenerative diseases or cancer. As the apoptotic potential of Tax is still debated, we addressed this question by testing the susceptibility of Tax(+) and Tax(-) murine fibroblasts to apoptosis under conditions of growth factor withdrawal or treatment with TNFalpha, which trigger apoptosis through different pathways, i.e., mitochondrial and receptor-mediated pathways, respectively. Results showed that Tax-expressing cells are protected from apoptotic death induced by serum deprivation but are sensitive to TNFalpha-mediated apoptosis, suggesting that Tax expression has different effects on cell death, depending on the apoptotic stimulus used. Analysis of the mechanism(s) involved in the resistance to serum depletion-induced apoptosis indicated that Tax(+) cells do not undergo release of cytochrome c from the mitochondrial intermembrane space or redistribution of Bax from the cytosol to mitochondria, two phenomena critical to the mitochondrial apoptotic pathway.

We studied the effect of protein phosphatase and kinase inhibitors on Tax-mediated transcription of constructs carrying the reporter gene chloramphenicol acetyl transferase under the control of either the full-length LTR of HTLV-I or three copies of the tax-responsive 21-bp repeats. We observed that treatment with okadaic acid, which inhibits the serine/threonine protein phosphatases type 1 and 2A, reduced HTLV-I LTR transcriptional activation in MT2 and K562 cells; on the contrary, the enhancer activity of the 21-bp sequences was significantly increased in both cell lines; treatment with the protein kinase C inhibitor H-7 blocked Tax-mediated transcription of both constructs. We also found that treatment with sodium orthovanadate, a tyrosine phosphatase inhibitor, reduced Tax-mediated activation of both plasmids. These findings indicated that specific serine/threonine phosphorylation events are required for Tax-mediated HTLV-I LTR activation and also suggested that phosphorylation at tyrosine residues is involved in this process.

Previous findings indicated that in vitro HTLV-I-infected cells are highly susceptible to spontaneous and chemically induced DNA-damage. To further study the role of different virus gene products in inducing chromosome abnormalities, MOLT-3 cells were transiently transfected with a tax expressing plasmid (pTax), and assayed for genetic damage by the micronucleus test. We found that pTax-transfected cells not only had a statistically higher baseline micronucleus value than non-transfected control cells, but also were more susceptible to Mitomycin C (MMC)-induced DNA damage. Furthermore, the use of human serum containing anti-kinetochore antibodies disclosed that tax enhances the clastogenic effect of MMC. No increase in total micronucleus frequency was observed when MMC treatment preceded pTax transfection, thus suggesting that the micronucleus increase might not be due to the additive effect of tax and MMC. These findings indicate that the viral tax protein could play an important role in inducing the chromosome damage frequently observed in HTLV-I-infected cells.

Sialated glycosphingolipids (gangliosides) were recently shown to induce internalisation of the CD4 Ag in lymphoid cells and dissociation of p56lck from CD4 (Repke et al. (1992) J. Immunol. 149, 2585-2591; Saggioro et al. (1993) J. Biol. Chem. 268, 1368-1375). The findings presented in this paper show that GM1 induces internalisation and the eventual degradation of the CD4 Ag also in the monocytic cell line U937. GM1 effects are independent of a possible activation of protein kinase C, as enzyme inhibitors which effectively blocked phorbol esters effects did not prevent GM1-induced CD4 internalisation and degradation. GM1 effects were also independent of a possible action on a CD4 associated kinase activity as we show that U937 cells lack any CD4-associated kinase activity.

The factors that regulate low viral expression and long latency after HTLV-I infection are poorly understood. To study the possible mechanisms involved in the regulation of gene expression and cell transformation, we studied whether (1) methylation could play a role in viral transcription, and (2) tax product could favor chromosomal instability. The results indicate that methylation of HTLV-I LTRs blocks their transcriptional activity and that tax protein triggers DNA damage.

Individuals infected with HTLV-1 harbor the virus mainly in CD4+ memory T-cells as a lifelong infection that remains subclinical in the majority of cases. However, about 3-5% of HTLV-1-infected individuals develop an aggressive T-cell neoplasia (ATLL) or a neurodegenerative disease (TSP/HAM) after a latency period ranging from years to decades. This review summarizes the current knowledge of the effects of the HTLV-1 proteins Tax, p13 and p12 on cell death and survival pathways. Tax, the major oncogenic determinant of HTLV-1, enhances cell survival through its effects on the NF-kappaB, CREB and AKT pathways and on the tumor suppressors p53 and Rb. p13 is targeted to the inner mitochondrial membrane and sensitizes cells to the Fas/ceramide apoptotic pathway and reactive oxygen species-mediated cell death. p12 enhances release of calcium from the endoplasmic reticulum and therefore may influence calcium-dependent apoptotic signals, including opening of the mitochondrial permeability transition pore. The long-term fate of HTLV-1-infected cells (apoptosis, survival, transformation) may therefore depend on the balance of the effects of Tax, p13 and p12 on cell death pathways.

Supernatants obtained from four HTLV-I transformed cell lines (MT2, MT4, C91/PL, and 81-66/45) induced in vitro migration of monocytes, polymorphonuclear leukocytes (PMN), and lymphocytes. The MT2, C91/PL, and 81-66/45 cell lines expressed both lymphotoxin (LT) and tumor necrosis factor (TNF-alpha) mRNA transcripts, and had TNF biological activity. In contrast, the MT4 cells did not express LT mRNA, had low levels of TNF-alpha transcript, and no TNF activity in the supernatant. Anti-TNF-alpha MAb, which blocks the chemotactic activity of recombinant TNF-alpha, had no inhibitory effect on the induction of migration by the MT2 and MT4 supernatants. Hence, no correlation was evident between TNF and chemotactic activity in supernatants of different HTLV-I-infected cell lines. Upon fractionation on Sephadex G50, the monocyte chemoattractant(s) eluted with two peaks in the 8-12 kD region, a size compatible with the chemotactic cytokines IL-8 and monocyte chemotactic protein (MCP). However, anti-IL-8 and anti-MCP antibodies did not have any effect, and Northern blot analysis showed that HTLV-I-transformed cell lines did not express mRNA transcripts of either IL-8 and MCP. These results demonstrate that HTLV-I transformed T-cell lines produce chemoattractant(s) active on PMN and monocytes, distinct from LT, TNF-alpha, IL-8, and MCP. Production of chemoattractants may play a role in the pathogenesis of diseases associated with HTLV-I infection.

HTLV-I infection is characterized by low viremia and restricted viral expression. While the mechanisms regulating viral latency are poorly understood, it is believed that interactions between viral and host cellular factor(s) are involved. Several lines of evidence indicate that HTLV-I provirus may be methylated in primary ATL leukemic cells. To determine whether methylation of the viral promoting sequences was sufficient to inhibit gene transcription, we methylated the HTLV-I LTR enzymatically at the HpaII (CCGG) sites. HTLV-I LTR contains several HpaII methylase-sensitive sites, and some involve one of the three 21-bp motifs, responsible for tax induction, as well as sequences that respond to phorbol 12-myristate 13-acetate (PMA). We found that CpG site-specific methylation of HTLV-I LTR sequences inhibits their transcriptional activation mediated by both tax product and PMA. This transcriptional block, however, was overcome when tax product and PMA were added together, thus indicating that tax and PMA act synergistically in bypassing the transcriptional block exerted by methylation.

The finding of dual HTLV-I and HIV-1 infection in populations at risk for AIDS raises the possibility that interaction between the two viruses might have clinical significance. It was shown that HTLV-I enhances HIV-1 expression, but whether HIV-1 activates HTLV-I remains to be demonstrated. To study HTLV-I behaviour following HIV-1 infection, we superinfected cells from two HTLV-I transformed cell lines with HIV-1 (strain IIIB). Viral RNA analysis indicated that HTLV-I expression in the doubly infected cells was moderately enhanced. Moreover, CAT assays in HTLV-I transformed cells transiently transfected with HTLV-I LTR-CAT disclosed higher activity in the HIV-1 superinfected cultures. This enhancement was observed only after infection with active HIV-1 virus, but not following exposure to inactivated viral particles or transfection with HIV-1 tat gene.

The frequency of spontaneous transformation, defined as the acquisition of the ability to form colonies in soft agar (anchorage independence) was compared in rat fibroblasts of recently established lines possessing pseudodiploid to pseudotetraploid numbers of chromosomes. Transformation frequency appeared to be independent on the ploidy level. The effect of colcemid on the transformation frequency of pseudodiploid fibroblasts was also examined. Colcemid treatment resulted in an increased frequency of transformation. In addition, most of the transformed clones, either derived from colcemid-treated cells, or derived from untreated cells, were characterized by significant changes in chromosome number. These results cannot be easily reconciled with the hypothesis that the main cause of spontaneous transformation is the appearance of recessive mutations. They rather support the idea that this conversion is the consequence of alterations in the number of certain chromosomes.

Chinese hamster lung cells cultured in the presence of crocidolite asbestos displayed inhibition of cell growth. Cell death was directly associated with the phagocytosis of larger fibres as observed with the aid of trypan blue. Watersoluble components of crocidolite did not appear to inhibit cell growth. Chromosomal aberrations were induced by the asbestos. The aberrations were confined mainly to structural aberrations — breaks and fragments. Electron-microscopic preparation indicated that asbestos was readily contained in phagosomes. Phagocytosed asbestos appeared to be a weak mutagen in its ability to induce gene mutation at the hypoxanthine-guanine phosphoribosyl transferase locus.

The presence of cell surface fibronectin was examined by indirect immunofluorescence in 8 groups of related rat fibroblast lines expressing an in vitro transformed phenotype. The transformed cells were selected for anchorage independent growth either after X-ray treatment (X-ray transformed cells) or from control cultures (spontaneously transformed cells). All transformed fibroblasts of the latter class showed reduced expression of fibronectin at the cell surface, whereas most of the X-ray transformed derivatives exhibited a fibronectin-positive phenotype, like the untransformed parents. Moreover, from the fibronectin-negative spontaneously transformed cells, ouabain-resistant variants were isolated, the majority of which had regained the capacity to form an extracellular matrix of fibronectin. These results emphasize the variability in the properties of transformed cells and suggest that the properties of in vitro transformed cells may depend on the cause of transformation.

Summary Although gold compounds are now recognized as promising anticancer agents, so far only gold(I) derivatives have been investigated for this purpose, whereas the use of gold(III) complexes has been hampered by their poor stability under physiological conditions. We have therefore carried out studies on selected gold(III) anticancer agents, showing enhanced stability due to the presence of chelating dithiocarbamato ligands. We found that they induce cancer cell death through both apoptotic and nonapoptotic mechanisms. They also inhibit thioredoxin reductase activity, generate free radicals, modify some mitochondrial functions, and increase ERK1/2 phosphorylation. Based on our results, we propose and discuss a working model suggesting that deregulation of the thioredoxin reductase/thioredoxin redox system is a major mechanism involved in the anticancer activity of the investigated gold(III)-dithiocarbamato complexes.

'Spontaneous' and mitomycin C (MMC)-induced sister chromatid exchanges (SCE) and chromatid breaks were scored in ANN-1 fibroblasts, a non-producer mouse cell line transformed by the Abelson murine leukemia virus (A-MuLV), a replication defective retrovirus whose genome contains the v-abl oncogene. Normal, non-transformed NIH3T3 fibroblasts were used as control. SCE and chromatid break frequencies in untreated or MMC-treated ANN-1 and NIH3T3 cells were compared with those observed in the same cells after infection with the helper murine Moloney leukemia virus (M-MuLV), which rescues the ability of A-MuLV to replicate in ANN-1 cells. The frequency of spontaneous and MMC-induced SCE were not significantly different in both ANN-1 and NIH3T3 cells, independently of M-MuLV infection. After M-MuLV infection, however, increased 'spontaneous' frequency of SCE and altered susceptibility to the induction of SCE by MMC was observed in both cell lines compared to M-MuLV-uninfected cells. In the case of chromatid breaks, the baseline frequency was not significantly different between the two cell lines both in the presence or in the absence of M-MuLV infection, nor was it significantly increased by M-MuLV, with respect to the value observed in uninfected cells. These results indicate that, at variance with what occurs with SCE, viral replication is not needed to increase the frequency of chromosomal aberrations and that the portion of A-MuLV genome alone is sufficient to increase chromatid breaks but not SCE in ANN-1 cells. Thus, in mouse cells carrying retroviruses, SCE and chromosomal aberrations seem to be independently generated, and influenced by different viral genes.