Arun Seth

ETS1, NFκB and AP1 synergistically transactivate the human GM-CSF promoter

Activation of helper T cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony stimulating factor (GMu2009–u2009CSF) is one such cytokine, whose increased expression results mostly from increases in transcription. Cis-acting elements with NFκB, AP1 and ETS-like binding motifs have been identified in the promoter region of the GMu2009–u2009CSF gene, and are important or essential for transcriptional activity following T cell activation. ETS1 is a transcription factor of the ETS family that is expressed in T cells. We have previously shown that ETS1 can transactivate GMu2009–u2009CSF in Jurkat T cells, but only after the cells have been stimulated by treatment with PMA and ionomycin, agents that mimic T cell activation. Thus we proposed that ETS1, which is expressed constitutively in Jurkat cells, may act in concert with PMA/ionomycin inducible factors. Here we show that ETS1 can transactivate a GMu2009–u2009CSF reporter construct in unstimulated Jurkat cells, providing that either NFκB or AP1 transcription factors are supplied by co-transfection. We confirm that binding of endogenous NFκB and AP1 is induced following PMA/ionomycin treatment of T cells. Transactivation by ETS1, NFκB and AP1 is synergistic, and mutation of the individual binding sites reveals that the transcriptional activities of these factors are inter-dependent. Our results suggest that constitutive ETS1, and inducible NFκB and AP1, cooperate as part of a higher order transcriptional complex in activated T cells.

Differential regulation of interstitial collagenase (MMP-1) gene expression by ETS transcription factors.

Expression of interstitial collagenase (MMP-1) has been detected in stromal fibroblasts of various malignant tumors. Here, we have studied the effect of three structurally different ETS transcription factors (ETS-1, ERGB/Fli-1, and PU.1) on MMP-1 promoter activity in NIH3T3 fibroblasts. ETS-1 increased the activity of 3.8u2009kb MMP-1 promoter construct up to tenfold, while ERGB/Fli-1 or PU.1 alone had no marked effect on basal promoter activity. ETS-1 also markedly potentiated enhancement of MMP-1 promoter by both c-Jun and JunB, whereas ERGB/Fli-1 augmented only the effect of c-Jun. Interestingly, PU.1 abolished induction of MMP-1 promoter by both c-Jun and JunB. Stimulation of MMP-1 promoter by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid was differentially augmented by ETS-1 and ERGB/Fli-1, and abrogated by PU.1. Co-transfection studies with MMP-1 promoter 5′-deletion constructs revealed that AP-1 site was necessary for PU.1-elicited suppression. As compared to control cell lines, PU.1-positive stable cells exhibited clearly weaker binding of c-Jun and JunD containing AP-1 complexes to MMP-1 promoter AP-1 element, as well as marked reduction in basal level and induction of c-jun mRNA by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid, suggesting a novel mechanism for PU.1-mediated inhibition of AP-1 dependent gene expression. These results show that three structurally distinct ETS transcription factors differently modulate AP-1 dependent upregulation of MMP-1 gene expression.

Down-regulation of T1A12/mac25, a novel insulin-like growth factor binding protein related gene, is associated with disease progression in breast carcinomas.

To define genes that are essential to the initiation and progression of breast cancer we utilized subtractive hybridization and differential display cloning techniques and isolated over 950 cDNAs from breast cell-lines derived from matched normal and tumor tissue. Of these, 102 cDNAs were characterized by DNA sequencing and Northern blot analysis. GenBank searches showed that one of these genes, T1A12 is identical to mac25, an insulin-like growth factor-binding protein related gene. Antibodies generated against the C-terminal region of the T1A12/mac25 protein were used to investigate its expression in 60 primary breast tissues. Sections of 12 benign, 16 ductal carcinoma in situ and 32 infiltrating ductal carcinoma specimens were examined. Strong immunoperoxidase staining was observed in luminal epithelial cells of normal lobules and ducts, in apocrine cells of cysts and fibroadenomas. Moderate to weak protein expression was found in hyperplastic and DCIS cells, but no specific staining was detected in invasive carcinoma cells. FISH mapping using a PAC clone localized the T1A12/mac25 gene to 4q12-13. Microsatellite length polymorphism was studied using markers for 4q in paired normal and tumor breast tissues. Thirty-three per cent (10/30) of the samples were found to be polymorphic with D4S189 and D4S231 microsatellite markers and LOH was detected in 50% (5/10) of these informative samples. Our data indicate that T1A12/mac25 expression is abrogated during breast cancer progression concomitant with loss of heterozygosity on chromosome 4q. T1A12/mac25 may therefore have a tumor suppressor-like function and its expression could indicate a disease with a more favorable status, having a better prognosis.

A novel transcription factor, ELF5, belongs to the ELF subfamily of ETS genes and maps to human chromosome 11p13–15, a region subject to LOH and rearrangement in human carcinoma cell lines

The ETS transcription factors are a large family implicated in the control of cellular proliferation and tumorigenesis. In addition, chromosomal translocations involving ETS family members are associated with a range of different human cancers. Given the extensive involvement of ETS factors in tumorigenesis, it becomes important to identify any additional ETS genes that may also play oncogenic roles. We identify a novel gene, ELF5, that appears to belong to the ELF (E74-like-factor) subfamily of the ETS transcription factor family, based upon similarity within the `ETS domain. ELF5 displays a similar, but more restricted, expression pattern to that of the newly isolated epithelium-specific ETS gene, ELF3. Unlike most other ETS family members, ELF5 is not expressed in hematopoietic compartments, but is restricted to organs such as lung, stomach, kidney, prostate, bladder and mammary gland. ELF5 is localized to human chromosome 11p13–15, a region that frequently undergoes loss of heterozygosity (LOH) in several types of carcinoma, including those of breast, kidney and prostate. We find that ELF5 expression is not detectable in a number of carcinoma cell lines, some of which display loss or rearrangement of an ELF5 allele. Similar to other ETS family members, ELF5 displays specific binding to DNA sequences containing a GGAA-core. In addition, ELF5 is able to transactivate through these ETS sequences, present upstream from a minimal promoter. Our data suggest that ELF5 may play roles in mammary, lung, prostate and/or kidney function, and possibly also in tumorigenesis.

Breast cancer genome anatomy: correlation of morphological changes in breast carcinomas with expression of the novel gene product Di12.

To determine which genes may be activated or inactivated during breast cancer development, we employed two cloning strategies (subtractive hybridization and differential display) using RNA samples from a human breast tumor and its matching normal breast cell line. Of 950 clones isolated, 102 cDNA inserts were analysed by DNA sequencing and database searching. We found 30 clones that were obviously unidentified, with no significant homology to any listed human gene. We focused upon one of the novel genes, Di12, that is differentially expressed as a 1.35u2009kb RNA in breast cancer tissues and cell-lines, and in several normal tissues. A full length cDNA of this gene was cloned, and its DNA sequence revealed an open reading frame of 339 amino acids. Antibodies to the ten N-terminal amino acids were developed to investigate the expression of Di12 in breast cancer cell-lines and tumors. The Di12 protein was found in tissue sections of infiltrating ductal carcinomas (IDCs), but not in benign or normal breast specimens. RTu2009–u2009PCR analysis confirmed expression of Di12 in 80% of infiltrating ductal carcinomas (IDCs). As IDC constitutes ∼70% of breast cancers seen clinically, the level of Di12 expression may be predictive of disease progression.

Regulation of the human stress response gene GADD153 expression: role of ETS1 and FLI-1 gene products

We have previously shown that ETS transcription factors, regulate cell growth and differentiation, and ETS1 and ETS2 are able to transcriptionally regulate wt p53 gene expression. In the present study we show that the ETS transcription factors also play a role in regulating expression of GADD153, a wt p53 inducible gene, which induces growth arrest and apoptosis in response to stress signals or DNA damage. We report the presence of a single EBS in the human GADD153 promoter, and that the GADD45 gene promoter lacks EBSs. The GADD153 promoter EBS shows a very high affinity for ETS1 and FLI-1 gene products. In addition, our data show that both ETS1 and FLI-1 strongly activate transcription of the GADD153 EBS linked to the CAT reporter gene. Our results also demonstrate how ETS1 and FLI-1 specifically regulate GADD153 expression. In addition, ectopic ETS2 protein expression resulted in only a weak induction of the same CAT reporter construct. The ETS1 and FLI-1 proteins provide a novel mechanism of activation for GADD153, allowing these two ETS genes to control its expression during cell growth and differentiation, rather than in response to oxidative stress.

Detection of squirrel monkey retroviral sequences in interferon samples

BACKGROUND/AIMSnInterferons have been used therapeutically in viral infections, and as immunomodulants in the treatment of different types of cancers. Interferons have been prepared from human lymphoid cell-lines, such as Namalwa, that contain integrated copies of squirrel monkey retrovirus proviral DNA. Squirrel monkey retrovirus is related to simian type D retroviruses, such as Mason-Pfizer monkey virus. Thus it is important to determine if these retroviral sequences are present in interferon preparations purified from human cell lines.nnnMETHODSnDNA samples were prepared from 75 commercial interferon preparations and analyzed for squirrel monkey retrovirus sequences by polymerase chain reaction and DNA sequencing. Since single polymerase chain reaction is not as sensitive, a nested polymerase chain reaction strategy was devised in order to detect squirrel monkey retrovirus-pol sequences. Amplification of beta-actin (human) sequences was used to confirm that samples contained human genomic DNA. To determine the authenticity of squirrel monkey retrovirus sequences, we analyzed amplified products by Southern blot hybridization and direct DNA sequencing.nnnRESULTS/CONCLUSIONSnThirty-nine samples were positive for squirrel monkey retrovirus-pol sequences by nested polymerase chain reaction. It is noteworthy that 29 samples were either weakly or very weakly positive by single polymerase chain reaction, thus stressing the importance of our sensitive polymerase chain reaction assay. However, it remains to be determined whether the residual DNA sequences detected by our sensitive nested polymerase chain reaction assay have biological consequences.