Jun and Fos family protein expression in human breast cancer: correlation of protein expression and clinicopathological parameters.

OBJECTIVES: The activator protein-1 (AP-1) is a dimeric transcription factor formed by members of the Jun and Fos protein family. AP-1 plays a role in a variety of physiological functions including cell proliferation and differentiation, although both c-Jun and c-Fos have also been implicated in oncogenic transformation and tumor progression. To further elucidate the role of AP-1 in breast cancer, we have investigated the expression of the AP-1 proteins c-Jun, JunB, JunD, phosphorylated c-Jun, c-Fos, Fral, Fra2 and the tumor supressor protein p53. METHODS: Protein expression was evaluated on a breast cancer tissue microarray with 58 lymph node positive or negative breast cancer specimens, 29 corresponding lymph node metastases, and 11 tissue samples from surrounding tumor-free tissue, each cored as triplicate. Jun and Fos protein family expression was evaluated by immunohistochemistry and was correlated with clinicopathological parameters. RESULTS: High expression levels were observed for c-Jun, JunD, c-Fos and Fra2, whereas JunB and Fral exhibited lower staining. c-Jun protein expression was correlated to Fral staining (p = 0.007, Kendall's Tau) and Fral was further associated with c-Fos (p < 0.001), JunD (p = 0.001) and Fra2 (p = 0.011) expression. JunD expression correlated with c-Fos (p < 0.001), JunB (p = 0.035) and c-Jun (p = 0.05). Activated c-Jun correlated with c-Fos expression (p = 0.041). JunB was negatively correlated to tumor stage, (p = 0.093, corr coeff. = -0.293, Spearman's correlation) but was significantly increased in nodal negative tumors (p = 0.004, Mann Whitney test). In addition, increased Fral expression showed a trend towards an increased overall survival (p = 0.077, RR = 0.534, Cox regression). CONCLUSION: Our results suggest an important role for JunB and Fral in the biological behavior of malignant breast tumors.

Novel t(5;9)(q33;q22) fuses ITK to SYK in unspecified peripheral T-cell lymphoma.

Among peripheral T-cell lymphomas (PTCL), the heterogeneous category of unspecified PTCL represents the most common subtype. Nevertheless, recurrent chromosomal translocations are unknown in this aggressive type of lymphoma. Here we describe a novel t(5;9)(q33;q22) in unspecified PTCL. Molecular analyses delineated the breakpoints to ITK and SYK resulting in a previously undescribed expression of the Syk tyrosine kinase by Itk. ITK-SYK transcripts were detected in five of 30 (17%) unspecified PTCL, but not in cases of angioimmunoblastic T-cell lymphoma (n=9) and anaplastic lymphoma kinase-negative anaplastic large-cell lymphoma (n=7). In all five translocation-positive cases, the breakpoints were identical fusing the N-terminal pleckstrin homology domain and proline-rich region of ITK to the tyrosine kinase domain of SYK. Three of the five t(5;9)(q33;q22)+ unspecified PTCL shared a very similar histological pattern with predominant involvement of lymphoid follicles and the same CD3+CD5+CD4+bcl-6+CD10+ immunophenotype. These results demonstrate the presence of a recurrent t(5;9)(q33;q22) in a subset of unspecified PTCL, which may represent a novel distinct subgroup of PTCL.

T(3;14)(p14.1;q32) involving IGH and FOXP1 is a novel recurrent chromosomal aberration in MALT lymphoma.

The three chromosomal translocations t(11;18)(q21;q21), t(14;18)(q32;q21), and t(1;14)(p22;q32) are associated with MALT lymphoma. In a case of MALT lymphoma of the thyroid, we observed t(3;14)(p14.1;q32) by cytogenetic analysis. Fluorescence in situ hybridization studies showed that the immunoglobulin heavy chain locus (IGH) was rearranged on chromosome 14. Long-distance inverse polymerase chain reaction identified FOXP1 as the partner gene on chromosome 3. To determine the frequency of the t(3;14)(p14.1;q32), two fluorescence in situ hybridization assays were established to screen 91 MALT lymphomas, all of which were negative for the above-mentioned three translocations, and eight splenic and six nodal marginal zone lymphomas. Overall, nine MALT lymphomas (10%) harbored t(3;14)(p14.1;q32) comprising tumors of the thyroid (three of six), ocular adnexa (four of 20), and skin (two of 20), whereas those of the stomach (n = 20), salivary gland (n = 20), and lung (n = 5) were negative as well as the splenic and nodal marginal zone lymphomas. Most t(3;14)(p14.1;q32) + MALT lymphomas harbored additional genetic abnormalities, such as trisomy 3. Further studies revealed that the three known translocations and t(3;14)(p14.1;q32) are mutually exclusive. Real-time quantitative reverse transcriptase polymerase chain reaction showed upregulation of FOXP1 in cases with t(3;14)(p14.1;q32) or trisomy 3. This study identifies FOXP1 as a new translocation partner of IGH in a site-dependent subset of MALT lymphomas.

The leukaemia-associated transcription factors EVI-1 and MDS1/EVI1 repress transcription and interact with histone deacetylase.

EVI-1 and its variant form, MDS1/EVI1, have been reported to act in an antagonistic manner and be differentially regulated in samples from patients with acute myeloid leukaemia and rearrangements of the long arm of chromosome 3. Here, we show that both EVI-1 and MDS1/EVI1 can repress transcription from a reporter construct containing EVI-1 binding sites and interact with histone deacetylase in mammalian cells. This interaction can be recapitulated in vitro and is mediated by a previously characterized transcription repression domain, whose activity is alleviated by the histone deacetylase inhibitor trichostatin A.

Transcription factor GATA-2 gene is located near 3q21 breakpoints in myeloid leukemia.

Rearrangements affecting chromosome band 3q21 are observed in a subgroup of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). However, little is known about the molecular consequences of such aberrations. We therefore established a PAC contig in the 3q21 breakpoint region and identified potential protein coding sequences by exon trapping. One of the exons isolated was from the human GATA-2 gene, which we showed to be transcribed from telomere to centromere. The majority of 3q21 breakpoints are located telomeric to the transcribed portion of this gene in a region that in mice appears to be necessary for proper promoter function. Results of GATA-2 expression analyses in leukemic cell lines as well as primary patient samples are compatible with the hypothesis that 3q21 aberrations contribute to leukemogenesis through deregulation of the hematopoietic transcription factor GATA-2.

Growth phase-regulated induction of Salmonella-induced macrophage apoptosis correlates with transient expression of SPI-1 genes.

Invasive Salmonella has been reported to induce apoptosis in a fraction of infected macrophages within 2 to 14 h from the time of infection by a mechanism involving the type III secretion machinery encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we show that bacteria in the transition from logarithmic to stationary phase cause 90% of the macrophages to undergo phagocytosis-independent, caspase-mediated apoptosis within 30 to 60 min of infection. The ability of Salmonella to induce this rapid apoptosis was growth phase regulated and cell type restricted, with epithelial cells being resistant. Apoptosis induction was also abrogated by disruption of the hilA gene (encoding a regulator of SPI-1 genes) and by the expression of a constitutively active PhoPQ. hilA itself and a subset of SPI-1 genes were transiently expressed during aerobic growth in liquid medium. Interestingly, however, hilA was found to be required only for the expression of the prgH gene, while sipB, invA, and invF were expressed in a hilA-independent manner. The expression of SPI-1 genes and the secretion of invasion-associated proteins correlated temporally with the induction of apoptosis and are likely to represent its molecular basis. Thus, growth phase transition regulates the expression and secretion of virulence determinants and represents the most efficient environmental cue for apoptosis induction reported to date.

Comparative expression analysis of the antagonistic transcription factors EVI1 and MDS1-EVI1 in murine tissues and during in vitro hematopoietic differentiation.

An alternative form of the transcription factor EVI1, MDS1-EVI1, which previously had been believed to exist only in the context of leukemic fusion mRNAs, has recently been shown to be expressed also in normal human tissues. Moreover, it acts as an antagonist of EVI1, activating transcription of reporter constructs repressed by EVI1. We cloned the murine homolog of MDS1-EVI1 as well as mMds1 and show localization of mMds1 close to mEvi1 on chromosome 3. Using RT-PCR, we demonstrate widespread expression of both Evi1 forms in the adult mouse, as well as their upregulation during in vitro hematopoietic differentiation. Our data underscore the biological importance of both EVI1 and MDS1-EVI1 and provide the basis for further studies of their function in the mouse model system.