Predictive markers in elderly patients with estrogen receptor-positive breast cancer treated with aromatase inhibitors: an array-based pharmacogenetic study.

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.

Genetic risk of subsequent esophageal cancer in lymphoma and breast cancer long-term survival patients: a pilot study.

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.

Block of a mitochondrial-mediated apoptotic pathway in Tax-expressing murine fibroblasts.

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.

Analysis of Tax-expressing cell lines generated from HTLV-I tax-transgenic mice: correlation between c-myc overexpression and neoplastic potential.

Human T-cell leukemia/lymphotropic virus type I (HTLV-I) infection causes a variety of human diseases, including adult T-cell leukemia/lymphoma. The viral transactivator Tax has been implicated as a key factor in the HTLV-I-induced transformation pathway. To investigate the components of this pathway, we derived fibroblast-like cell lines, designated T6 and T9, from tail biopsies of tax-transgenic C57BL/6 mice that do not develop tumors. Phenotypic characterization of T6 and T9 cells and T6-derived subclones revealed that they differ in their abilities to form foci in vitro and tumors in vivo. The observed differences in the levels of Tax expression did not correlate with their degree of neoplastic potential. However, a control cell line derived from a nontransgenic C57BL/6 mouse did not form foci in vitro or tumors in vivo, indicating that Tax was required for the transformation process. Results of Northern analyses showed that the T9 cells and the highly malignant derivatives of T6 cells expressed elevated levels of c-myc mRNA. These findings suggest that progression of the tax-transgenic cells toward a more malignant phenotype might involve c-myc deregulation.

Inflammatory polyarthropathy and bone remodeling in HTLV-I Tax-transgenic mice.

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.

Identification of MIP-1 alpha/LD78 as a monocyte chemoattractant released by the HTLV-I-transformed cell line MT4.

It is known that the HTLV-I-transformed cell line MT4 releases chemotactic activity for monocytes spontaneously. The MT4 monocyte chemoattractant was purified to homogeneity and sequencing of 25 amino acids revealed identity with the C-C chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha/LD78). An anti-MIP-1 alpha/LD78 rabbit antiserum substantially inhibited chemotaxis of the MT4 chemoattractant. MT4 cells constitutively expressed MIP-1 alpha/LD78 but not the C-C chemokines MCP-1, RANTES, and MIP-1 beta/Act2 and the C-X-C chemokines IL-8, gro alpha, and gro beta. MT4-derived MIP-1 alpha/LD78 was active on monocytes but was a weak chemoattractant for polymorphonuclear leukocytes. Thus, MIP-1 alpha/LD78 is a major monocyte chemoattractant released by HTLV-I-transformed T cells. Expression of MIP-1 alpha/LD78, a leukocyte chemotactic and myelosuppressive molecule, may play an important role in the manifestations of HTLV-I-related diseases.

Tax-induced HTLV-I LTR transcriptional activation is modulated by phosphorylation.

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.

Tax protein of human T-lymphotropic virus type I triggers DNA damage.

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.

The monosialoganglioside GM1 induces internalisation and degradation of the CD4 antigen in U937 cells: evidence for a novel mechanism of CD4 down-modulation in a p56lck-negative cell line, which is independent of protein kinase C activation.

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.

Mechanism of action of the monosialoganglioside GM1 as a modulator of CD4 expression. Evidence that GM1-CD4 interaction triggers dissociation of p56lck from CD4, and CD4 internalization and degradation.

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.

Studies on the mechanisms of HTLV-I leukemogenesis.

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.

Chemoattractant(s) in culture supernatants of HTLV-I-Infected T-cell lines.

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.

Transcriptional block of HTLV-I LTR by sequence-specific methylation.

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.

Reciprocal activation of human T-lymphotropic viruses in HTLV-I-transformed cells superinfected with HIV-1.

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.

Human T-lymphotropic virus type I transcriptional regulation by methylation.

While human T-lymphotropic virus type I (HTLV-I) proviral genome is readily detected in leukemic lymphocytes from adult T-cell leukemia patients, viral antigens or viral RNA are not expressed unless these cells are cultured. To address the problem of possible restriction mechanism of HTLV-I replication, we studied the methylation state of provirus in four HTLV-I transformed cell lines. These cell lines are chronically infected with HTLV-I and carry several copies of proviruses but are characterized by different viral expression. Molecular analysis showed that in MT4 cell line, which expresses a low level of viral RNA and proteins, not only are the HTLV-I proviruses heavily methylated but methylation also involves large regions around and within the long terminal repeats. MT4 cells treatment with 5-azacytidine led to an increase in both total viral RNA and p24 gag protein expression. These findings indicate that proviral methylation is responsible for the low viral expression in MT4 cells and also suggest that this phenomenon may be relevant to HTLV-I in vivo latency.

Relationship between DNA replicon size and SCE induction in BALB/c and BALB/Mo mouse lymphocytes.

We studied the DNA replicon size in BALB/c and BALB/Mo mouse lymphocytes by the method of bromodeoxyuridine photolysis. After treatment of the BALB/Mo lymphocytes in vitro with mitomycin C, the average DNA replicon size appeared to be significantly smaller than that observed in BALB/c lymphocytes treated similarly. In these conditions an increased susceptibility to SCE induction in BALB/Mo lymphocytes had been observed. In the presence of both mitomycin C and cordycepin (an antiviral drug), both the DNA replicon size and the SCE frequency returned to normal values.

In vitro immune-receptor gene rearrangements in clonal thymic lymphomas induced by Abelson murine leukemia virus.

The Abelson and Moloney murine leukemia virus complex (A-MuLV/M-MuLV) induces rapidly growing thymic lymphomas following direct injection into the thymus of newborn BALB/c and C57BL/6 mice. Southern blot analysis with a v-abl specific probe not only demonstrated that primary tumors are clonal, but also that the pattern of A-MuLV provirus integration is quite stable in primary tumor cells, as well as in their derived cell lines and clones. Most of the cell samples were able to rearrange the immunoglobulin heavy chain genes in culture, whereas in two cases the T cell receptor gamma chain genes also underwent rearrangement. Since the recombination mechanism is operative only in very immature lymphoid cells, these data provide indirect evidence for the lack of differentiation of A-MuLV cell targets in the thymus.

Chromosome damage induced in cord blood T-lymphocytes infected in vitro by HTLV-I.

Experiments were carried out to investigate whether the human T-lymphotropic virus type I (HTLV-I), alone or in combination with a chemical mutagen such as mitomycin C (MMC), has the capacity to damage host chromosomes. Cord-blood T lymphocytes (CBL) were infected by co-cultivation with lethally irradiated HTLV-I-producing cells. Infected and immortalized CBL were then studied for frequencies of sister chromatid exchanges (SCE), chromosome breaks and micronuclei. HTLV-I-infected cells had statistically higher baseline SCE, chromosome aberrations and micronucleus values than the uninfected control CBL. While MMC treatment further augmented these values both in control and in infected lymphocytes, the latter did not show dose-related increases, most likely because of the more pronounced MMC-induced delaying effect on cell progression to mitosis. In view of similar previous observations in mouse lymphocytes carrying the Moloney murine leukemia virus, it is suggested that expression of a common retrovirus gene product, such as the pol endonuclease, might be responsible for the cytogenetic abnormalities observed. In addition to the IL-2 autocrine loop, the direct induction of chromosome damage by HTLV-I in target lymphocytes may be related to the pathogenesis of malignancies associated with HTLV-I infection.

In vitro malignant progression of cells derived from Abelson murine leukaemia virus-induced thymic lymphomas.

Cell lines derived from A-MuLV induced thymic lymphomas in BALB/c and C57BL/6 mice were analysed for their in vivo and in vitro potential of growth. Despite their immunogenicity, cell lines of BALB/c origin readily grew in syngeneic recipients. On the contrary, all cell lines of C57BL/6 origin failed to grow in immunocompetent hosts even though they were able to form tumours in immunosuppressed syngeneic mice. Among C57BL/6 lymphoma cells progression toward a more malignant phenotype was observed in TB6-3 cells, and in their derived clones, after several in vitro passages. This event was accompanied by the in vitro loss of requirement for exogenous growth factor(s) when tumorigenic TB6-3 cells were plated at high density. Moreover, culture medium from fully malignant TB-3 cells was mitogenic for mature T-lymphoma cells suggesting the involvement of an autocrine mechanism in the control of cell proliferation. Apparently, the viral oncogene (v-abl) is not directly involved in malignant progression since no differences between nontumorigenic and tumorigenic cells could be detected in A-MuLV integration patterns, v-abl specific mRNA expression, and P160gag-abl production.

Sister chromatid exchanges and chromosomal aberrations in mouse cells infected with the Abelson and Moloney leukemia viruses.

'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.