Ada Sacchi

Physical and Functional Interaction between p53 Mutants and Different Isoforms of p73

p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro andin vivo with p73α, β, γ, and δ. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.

Induction of hTERT Expression and Telomerase Activity by Estrogens in Human Ovary Epithelium Cells

ABSTRACT In mammals, molecular mechanisms and factors involved in the tight regulation of telomerase expression and activity are still largely undefined. In this study, we provide evidence for a role of estrogens and their receptors in the transcriptional regulation of hTERT, the catalytic subunit of human telomerase and, consequently, in the activation of the enzyme. Through a computer analysis of the hTERT 5′-flanking sequences, we identified a putative estrogen response element (ERE) which was capable of binding in vitro human estrogen receptor α (ERα). In vivo DNA footprinting revealed specific modifications of the ERE region in ERα-positive but not ERα-negative cells upon treatment with 17β-estradiol (E2), indicative of estrogen-dependent chromatin remodelling. In the presence of E2, transient expression of ERα but not ERβ remarkably increased hTERT promoter activity, and mutation of the ERE significantly reduced this effect. No telomerase activity was detected in human ovary epithelial cells grown in the absence of E2, but the addition of the hormone induced the enzyme within 3 h of treatment. The expression of hTERT mRNA and protein was induced in parallel with enzymatic activity. This prompt estrogen modulation of telomerase activity substantiates estrogen-dependent transcriptional regulation of the hTERT gene. The identification of hTERT as a target of estrogens represents a novel finding which advances the understanding of telomerase regulation in hormone-dependent cells and has implications for a potential role of hormones in their senescence and malignant conversion.

Mutant p53: an oncogenic transcription factor.

Inactivation of tumor-suppressor genes is one of the key hallmarks of a tumor. Unlike other tumor-suppressor genes, p53 is inactivated by missense mutations in half of all human cancers. It has become increasingly clear that the resulting mutant p53 proteins do not represent only the mere loss of wild-type p53 tumor suppressor activity, but gain new oncogenic properties favoring the insurgence, the maintenance, the spreading and the chemoresistance of malignant tumors. The actual challenge is the fine deciphering of the molecular mechanisms underlying the gain of function of mutant p53 proteins. In this review, we will focus mainly on the transcriptional activity of mutant p53 proteins as one of the potential molecular mechanisms. To date, the related knowledge is still quite scarce and many of the raised questions of this review are yet unanswered.

Mutant p53 gain of function: reduction of tumor malignancy of human cancer cell lines through abrogation of mutant p53 expression

Mutations in the TP53 tumor suppressor gene are the most frequent genetic alteration in human cancers. These alterations are mostly missense point mutations that cluster in the DNA binding domain. There is growing evidence that many of these mutations generate mutant p53 proteins that have acquired new biochemical and biological properties. Through this gain of function activity, mutant p53 is believed to contribute to tumor malignancy. The purpose of our study was to explore mutant p53 as a target for novel anticancer treatments. To this aim, we inhibited mutant p53 expression by RNA interference in three different cancer cell lines endogenously expressing mutant p53 proteins, and evaluated the effects on the biological activities through which mutant p53 exerts gain of function. We found that depletion of mutant p53 reduces cell proliferation, in vitro and in vivo tumorigenicity, and resistance to anticancer drugs. Our results demonstrate that mutant p53 knocking down weakens the aggressiveness of human cancer cells, and provides further insight into the comprehension of mutant p53 gain of function activity in human tumor.

p53 re-expression inhibits proliferation and restores differentiation of human thyroid anaplastic carcinoma cells

Alterations of the tumor suppressor gene p53 are uncommon in differentiated thyroid neoplasia but are detected at high frequency in anaplastic thyroid carcinoma suggesting that impaired p53 function may contribute to the undifferentiated and highly aggressive phenotype of these tumors. Effects of wild type p53 (wt-p53) re-expression were investigated in a human anaplastic thyroid carcinoma cell line (ARO) expressing a mutated p53. ARO cells were stably transfected with the temperature-sensitive p53 Val135 gene (ts-p53) which exhibits wild type-like activity at 32°C. Exogenous wt-p53 function in ARO-tsp53 clones was assessed by evaluating its transcriptional activity on a CAT reporter vector containing p53 binding sites. At 32°C, a significant reduction in the proliferation rate (≈percnt;50%) was observed, with accumulation of cells in the G0/G1 phase of the cell cycle. This effect was accompanied by induction of the expression of the growth inhibitor p21/Waf1 gene. At 32°C, ARO-tsp53 clones also showed a marked impairment of their tumorigenic potential. Furthermore, transfected clones re-acquired the ability to respond to thyrotropin (TSH) stimulation showing an increased expression of thyroid-specific genes (thyroglobulin, thyroperoxidase and TSH receptor). In conclusion, re-expression of wt-p53 activity in ARO cells, inhibits cell proliferation and restores responsiveness to physiological stimuli.

The insulin-like growth factor I receptor as a physiologically relevant target of p53 in apoptosis caused by interleukin-3 withdrawal.

The wild-type p53 protein is known to modulate apoptosis induced in 32D murine hemopoietic cells by interleukin-3 withdrawal. In 32D cells and in 32D cells constitutively expressing a temperature-sensitive mutant of p53 (32Dtsp53), overexpression of a wild-type (but not a mutant) insulin-like growth factor I receptor (IGF-IR) protects these cells from apoptosis. A tsp53 in its wild-type conformation causes a decrease in the levels of IGF-IRs, and this decrease is accompanied by increased sensitivity of these cells to apoptosis. However, when the expression of the IGF-IR cDNA is regulated by a viral promoter, IGF-IR levels are not decreased by a wild-type p53, and apoptosis does not occur. These findings show that, in 32Dtsp53 cells, the IGF-IR is a physiologically relevant target of p53 in the process of apoptosis.

Growth and Differentiation Signals by the Insulin-like Growth Factor 1 Receptor in Hemopoietic Cells Are Mediated through Different Pathways

The type 1 insulin-like growth factor receptor (IGF-IR) plays an important role in the growth of cells both in vivo and in vitro. The IGF-IR is also capable of inducing differentiation in a number of cell types, raising the question of how the same receptor can send two seemingly contradictory signals, one for growth and one for differentiation. Using 32D cells, which are murine hemopoietic cells, we show that the activated IGF-IR can induce differentiation along the granulocytic pathway in a manner similar to the granulocyte colony-stimulating factor. We find that one of the major substrates of the IGF-IR, the insulin receptor substrate-1 inhibits IGF-I-mediated differentiation of 32D cells. In the absence of insulin receptor substrate-1, functional impairment of another major substrate of the IGF-IR, the Shc proteins, is associated with a decrease in the extent of differentiation. Although the end points of the respective pathways remain to be defined, these results show for the first time that IGF-I-mediated growth or differentiation of hemopoietic cells may depend on a balance between two of its substrates.

Inhibition of HIF-1alpha activity by homeodomain-interacting protein kinase-2 correlates with sensitization of chemoresistant cells to undergo apoptosis

BackgroundHomeodomain-interacting protein kinase-2 (HIPK2), a transcriptional co-repressor with apoptotic function, can affect hypoxia-inducible factor 1 (HIF-1) transcriptional activity, through downmodulation of its HIF-1α subunit, in normoxic condition. Under hypoxia, a condition often found in solid tumors, HIF-1α is activated to induce target genes involved in chemoresistance, inhibition of apoptosis and tumor progression. Here, we investigated whether the HIPK2 overexpression could downregulate HIF-1α expression and activity in tumor cells treated with hypoxia-mimicking condition, and evaluated whether HIPK2-dependent downregulation of HIF-1α could sensitize chemoresistant tumor cells to adriamycin (ADR)-induced apoptosis.MethodsTumor cell lines carrying wild-type p53, siRNA p53, or mutant p53 were overexpressed with HIPK2 (full length or catalytic inactive mutant) and treated with cobalt chloride (CoCl2) to mimic hypoxia, in the presence or absence of ADR treatment. HIF-1α expression was measured by semiquantitative reverse-transcriptase (RT)-PCR and Western immunoblotting and HIF-1 activity was evaluated by luciferase assay using reporter plasmid containing hypoxia response elements (HREs) upstream of luciferase gene. HIF-1 target genes, including multidrug resistance 1 (MDR1) and the antiapoptotic Bcl2 were determined by RT-PCR. Cell survival and apoptosis were measured by colony assay and cleavage of the caspase-3 substrate PARP, respectively.ResultsOverexpression of HIPK2 resulted in downmodulation of cobalt-stabilized HIF-1α protein and HIF-1α mRNA levels, with subsequent inhibition of HIF-1 transcriptional activity. MDR1 and Bcl-2 gene expression was downmodulated by HIPK2 overexpression in cobalt-treated cells. Inhibition of HIF-1 transcriptional activity was dependent on HIPK2 catalytic activity. HIPK2 overexpression did not induce per se apoptosis of cobalt-treated cells, on the contrary it sensitized cobalt-treated cells to ADR-induced apoptosis, regardless of their p53 status.ConclusionThe ability of HIPK2 to restore the apoptosis-inducing potential of chemotherapeutic drug in hypoxia-mimicking condition and therefore to sensitize chemoresistant tumor cells suggests that HIPK2 may induce fundamental alterations in cell signaling pathways, involving or not p53 function. Thus potential use of HIPK2 is promising for cancer treatment by potentiating cytotoxic therapies, regardless of p53 cell status.

The Transcriptional Repressor ZEB Regulates p73 Expression at the Crossroad between Proliferation and Differentiation

ABSTRACT The newly discovered p73 gene encodes a nuclear protein that has high homology with p53. Furthermore, ectopic expression of p73 in p53+/+ and p53−/− cancer cells recapitulates some of the biological activities of p53 such as growth arrest, apoptosis, and differentiation. p73−/−-deficient mice exhibit severe defects in proper development of the central nervous system and pheromone sensory pathway. They also suffer from inflammation and infections. Here we studied the transcriptional regulation of p73 at the crossroad between proliferation and differentiation. p73 mRNA is undetectable in proliferating C2C12 cells and is expressed at very low levels in undifferentiated P19 and HL60 cells. Conversely, it is upregulated during muscle and neuronal differentiation as well as in response to tetradecanoyl phorbol acetate-induced monocytic differentiation of HL60 cells. We identified a 1-kb regulatory fragment located within the first intron of p73, which is positioned immediately upstream to the ATG codon of the second exon. This fragment exerts silencer activity on p73 as well as on heterologous promoters. The p73 intronic fragment contains six consensus binding sites for transcriptional repressor ZEB, which binds these sites in vitro and in vivo. Ectopic expression of dominant-negative ZEB (ZEB-DB) restores p73 expression in proliferating C2C12 and P19 cells. Thus, transcriptional repression of p73 expression by ZEB binding may contribute to the modulation of p73 expression during differentiation.

Insulin receptor substrate-1, p70S6K and cell size in transformation and differentiation of Hemopoietic cells

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70S6K, which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70S6K and an increase in cell size. Phosphorylationin vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70S6K seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70S6K in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70S6K activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.