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The Kruppel-like factor 5 (KLF5) is a transcription factor that regulates cellular signaling involved in cell proliferation and oncogenesis. Here, we report that KLF5 interacts with tumor suppressor p53 in regulating the expression of the inhibitor-of-apoptosis protein survivin, which may play a role in pathological process of cancer. The core promoter region of survivin contains multiple GT-boxes that have been characterized as KLF5 response elements. Deletion and mutation analyses as well as chromatin immunoprecipitation and electronic mobility shift assay indicated that KLF5 binds to the core survivin promoter and strongly induces its activity. Furthermore, we demonstrated that KLF5 protein is able to bind to p53 and abrogate the p53-regulated repression of survivin. Transfection of KLF5 into a KLF5-negative acute lymphoblastic leukemia cell line EU-8 enhanced survivin expression, and conversely, silencing of KLF5 by small interfering RNA in a KLF5-overexpressing acute lymphoblastic leukemia cell line EU-4 down-regulated survivin expression. The KLF5 small interfering RNA-mediated down-regulation of survivin sensitized EU-4 cells to apoptosis induced by chemotherapeutic drug doxorubicin. These findings identify a novel regulatory pathway for the expression of survivin under the control of KLF5 and p53. Deregulation of this pathway may result in overexpression of survivin in cancer, thus contributing to drug resistance.

The eukaryotic initiation factor 4E (eIF4E) plays important roles in transformation and cancer progression. It is frequently overexpressed in malignant cells, one mechanism of which is through transcriptional activation by c-myc. Here, we report that high level of eIF4E expression and its tumorigenicity could be alternatively associated with defects of p53, since we found that induction of wt-p53 repressed eIF4E expression. Gene transfection of p53 inhibited eIF4E promoter activity, while inactivation of p53 either by mutation or by over-expression of MDM2 resulted in stimulation of eIF4E promoter activity. We demonstrated that p53-repression of eIF4E was regulated by c-myc. The wt-p53 can physically bind to c-myc, which inhibited binding of c-myc to eIF4E promoter and c-myc-stimulated promoter activity. These results suggest that the expression of eIF4E is reciprocally regulated by p53 and c-myc, and loss of p53-mediated control over c-myc-dependent transactivation of eIF4E may represent a novel mechanism for eIF4E-mediated neoplastic transformation and cancer progression.

IKKalpha, a subunit of IkBalpha kinase (IKK) complex, has an important role in the activation of nuclear factor-kB (NF-kB), a key regulator of normal and tumor cell proliferation, apoptosis, and response to chemotherapy. However, little is known about the transcriptional regulation of the IKKalpha gene itself. The present study revealed that the transcriptional induction of the IKKalpha gene is positively regulated by binding ETS-1, the protein product of the ETS-1 proto-oncogene. Furthermore, ETS-1 mediated activation of IKKalpha is negatively regulated by p53 binding to ETS-1. By analyzing the genomic DNA sequence, we identified the putative IKKalpha promoter sequence in the 5'-flanking untranslated region of the IKKalpha gene. Transfection of EU-4, an acute lymphoblastic leukemia (ALL) cell line, with plasmids containing the IKKalpha 5'-untranslated region sequence upstream of the luciferase reporter showed that this region possessed major promoter activity. Induction or enforced overexpression of p53 represses IKKalpha mRNA and protein expression as well as IKKalpha promoter activity. Deletion and mutation analyses as well as chromatin immunoprecipitation and electrophoretic mobility shift assay indicated that ETS-1 binds to the core IKKalpha promoter and strongly induces its activity. Although p53 does not directly bind to the IKKalpha promoter, it physically interacts with ETS-1 and specifically inhibits ETS-1-induced IKKalpha promoter activity. These results suggest that the proximal 5'-flanking region of the IKKalpha gene contains a functional promoter reciprocally regulated by p53 and ETS-1. Furthermore, loss of p53-mediated control over ETS-1-dependent transactivation of IKKalpha may represent a novel pathway for the constitutive activation of NF-kB-mediated gene expression and therapy resistance in cancer.

Survivin is a unique member of the inhibitor of apoptosis protein family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DeltaEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild-type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53-143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53-143. When EU-4/p53-143 cell culture was shifted from 37.5 degrees C to the wt-p53-permissive temperature (32.5 degrees C), the expression of survivin and survivin-DeltaEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a proapoptotic factor and its expression is upregulated by p53.

To investigate the possible role of inhibiting NF-kB activation in sensitizing tumor cells to chemotherapy-induced apoptosis, we transfected the dominant-negative mutant inhibitor of NF-kB (IkBm) into the EU-1 cell line, an acute lymphoblastic leukemia (ALL) line with constitutive NF-kB activation. Overexpression of IkBm significantly reduced constitutive NF-kB activity in EU-1 cells, resulting in decreased cell growth. In response to apoptosis induced by chemotherapeutic drugs, IkBm-transfected cells (EU-1/IkBm) exhibited increased sensitivity to vincristine (VCR), whereas sensitivity to doxorubicin (Dox) was not changed as compared to neo-transfected control (EU-1/neo) cells. To further evaluate the link between IkBm and sensitivity to Dox and VCR, we demonstrated that both endogenous IkBalpha and ectopic IkBm bind to p53. In response to Dox, the cytosolic p53.IkBalpha complex rapidly dissociated due to downregulation of IkBalpha. However, the p53.IkBm complex did not dissociate under these conditions. Although treatment of EU-1/IkBm cells with Dox increased the expression of p53, the nondissociating p53.IkBm complex resulted in decreased p53 function, as demonstrated by absence of cell-cycle arrest and induction of p53 target genes. Contrastingly, VCR-induced cell death neither downregulated IkBalpha nor induced p53, as shown by the lack of NF-kB activation and p53-mediated gene expression in VCR-treated cells. Our data suggest that IkBm simultaneously downregulates NF-kB activation and sequesters p53 in the cytoplasm, thus enhancing NF-kB-regulated apoptosis but blocking p53-dependent apoptosis.