MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is known for its very poor overall prognosis. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. We used 377 feature microRNA (miRNA) arrays to investigate miRNA expression in normal pancreas, chronic pancreatitis, and PDAC tissues as well as PDAC-derived cell lines. A pancreatic miRNome was established comparing the data from normal pancreas with a reference set of 33 human tissues. The expression of miR-216 and -217 and lack of expression of miR-133a were identified as characteristic of pancreas tissue. Unsupervised clustering showed that the three pancreatic tissues types can be classified according to their respective miRNA expression profiles. We identified 26 miRNAs most prominently misregulated in PDAC and a relative quantitative reverse transcriptase-polymerase chain reaction index using only miR-217 and -196a was found to discriminate normal pancreas, chronic pancreatitis and cancerous tissues, establishing a potential utility for miRNAs in diagnostic procedures. Lastly, comparing differentially expressed genes from PDAC with predicted miRNA target genes for the top 26 miRNAs, we identified potential novel links between aberrant miRNA expression and known target genes relevant to PDAC biology. Our data provides novel insights into the miRNA-driven pathophysiological mechanisms involved in PDAC development and offers new candidate targets to be exploited both for diagnostic and therapeutic strategies.

Characterization of the oligomerization defects of two p53 mutants found in families with Li-Fraumeni and Li-Fraumeni-like syndrome.

Recently two germline mutations in the oligomerization domain of p53 have been identified in patients with Li-Fraumeni and Li-Fraumeni-like Syndromes. We have used biophysical and biochemical methods to characterize these two mutants in order to better understand their functional defects and the role of the p53 oligomerization domain (residues 325-355) in oncogenesis. We find that residues 310-360 of the L344P mutant are monomeric, apparently unfolded and cannot interact with wild-type (WT) p53. The full length L344P protein is unable to bind sequence specifically to DNA and is therefore an inactive, but not a dominant negative mutant. R337C, on the other hand, can form dimers and tetramers, can hetero-oligomerize with WTp53 and can bind to a p53 consensus element. However, the thermal stability of R337C is much lower than that of WTp53 and at physiological temperatures more than half of this mutant is less than tetrameric. Thus, the R337C mutant retains some functional activity yet leads to a predisposition to cancer, suggesting that even partial inactivation of p53 oligomerization is sufficient for accelerated tumour progression.

Structure and functionality of a designed p53 dimer.

P53 is a homotetrameric tumor suppressor protein involved in transcriptional control of genes that regulate cell proliferation and death. In order to probe the role that oligomerization plays in this capacity, we have previously designed and characterized a series of p53 proteins with altered oligomeric states through hydrophilc substitution of residues Met340 or Leu344 in the normally tetrameric oligomerization domain. Although such mutations have little effect on the overall secondary structural content of the oligomerization domain, both solubility and the resistance to thermal denaturation are substantially reduced relative to that of the wild-type domain. Here, we report the design and characterization of a double-mutant p53 with alterations of residues at positions Met340 and Leu344. The double-mutations Met340Glu/Leu344Lys and Met340Gln/Leu344Arg resulted in distinct dimeric forms of the protein. Furthermore, we have verified by NMR structure determination that the double-mutant Met340Gln/Leu344Arg is essentially a "half-tetramer". Analysis of the in vivo activities of full-length p53 oligomeric mutants reveals that while cell-cycle arrest requires tetrameric p53, transcriptional transactivation activity of monomers and dimers retain roughly background and half of the wild-type activity, respectively.

Mode of pulsatile follicle-stimulating hormone secretion in gonadal hormone-sufficient and -deficient women--a clinical research center study.

To test the hypothesis that FSH is secreted at least in part within discrete secretory bursts in women and that the characteristics of episodic FSH secretion are altered within differing gonadal hormone environments, we measured FSH by immunoradiometric assay every 10 min for 24 h in premenopausal women during the early follicular (EF), late follicular (LF), and midluteal (ML) phases of the menstrual cycle and in postmenopausal (PM) women (n = 8 in each group). Secretory events were evaluated using multiparameter deconvolution. FSH was secreted in an episodic manner, with the number of secretory bursts (per 24 h; mean +/- SEM) detected in LF (20 +/- 0.79) and PM (20 +/- 0.90) women being greater than that in EF (16 +/- 0.88) and ML (14 +/- 0.93) women. FSH secretory burst mass (milliinternational units per mL) was significantly higher in PM (12 +/- 1.6) than in EF (1.8 +/- 0.21), LF (3.1 +/- 1.3), or ML (0.8 +/- 0.11) women and primarily reflected a relative increase in the maximal secretory rate rather than increased burst half-duration. The estimated half-life (minutes) of endogenous FSH in LF women (155 +/- 18) was shorter than those calculated in EF (251 +/- 24), ML (277 +/- 38), and PM (231 +/- 18) women. Cross-correlation analysis showed strongly positive associations between successively paired serum FSH and LH concentrations in all four groups of women. Deconvolution of simultaneously obtained LH concentration-time series revealed statistically significant concordance (13-25%) between FSH and LH secretory episodes at a lag time of 0 min in EF, LF, and PM women and when LH secretory bursts led FSH secretory bursts by 10 min in ML phase women. However, as 75-87% of FSH and LH secretory pulses were discordant, we infer the operation of distinct control mechanisms in the generation of FSH and LH release episodes. In summary, these results suggest that FSH is secreted within discrete secretory bursts in women, that the mass and frequency of FSH secretory bursts differ in women exhibiting various gonadal hormone environments, and that FSH and LH secretory bursts occur coincidentally at a higher rate than expected on the basis of chance alone, but at such a low overall rate of concordance that distinct mechanisms probably operate to direct episodic FSH and LH secretory activity.

p73 and p63 are homotetramers capable of weak heterotypic interactions with each other but not with p53.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human cancers. Recent identification of two human homologues of p53 has raised the prospect of functional interactions between family members via a conserved oligomerization domain. Here we report in vitro and in vivo analysis of homo- and hetero-oligomerization of p53 and its homologues, p63 and p73. The oligomerization domains of p63 and p73 can independently fold into stable homotetramers, as previously observed for p53. However, the oligomerization domain of p53 does not associate with that of either p73 or p63, even when p53 is in 15-fold excess. On the other hand, the oligomerization domains of p63 and p73 are able to weakly associate with one another in vitro. In vivo co-transfection assays of the ability of p53 and its homologues to activate reporter genes showed that a DNA-binding mutant of p53 was not able to act in a dominant negative manner over wild-type p73 or p63 but that a p73 mutant could inhibit the activity of wild-type p63. These data suggest that mutant p53 in cancer cells will not interact with endogenous or exogenous p63 or p73 via their respective oligomerization domains. It also establishes that the multiple isoforms of p63 as well as those of p73 are capable of interacting via their common oligomerization domain.

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53.

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer-dimer interface of the tetrameric oligomerization domain of p53 (residues 325-355). Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state (tetramer, dimer, or equilibrium mixture of both) of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met (tetramer) > Ser Asp, His, Gln, > Glu, Lys (dimer), whereas the experimental results showed the following order: Met (tetramer) > Ser > Gln > His, Lys > Asp, Glu (dimers). For residue 344, the calculated trend was Leu (tetramer) > Ala > Arg, Gln, Lys (dimer), and the experimental trend was Leu (tetramer) > Ala, Arg, Gln, Lys (dimer). The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the alpha-helices at the dimer-dimer interface. Overall, this initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.

ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase.

Telomere loss has been proposed as a mechanism for counting cell divisions during aging in normal somatic cells. How such a mitotic clock initiates the intracellular signalling events that culminate in G1 cell cycle arrest and senescence to restrict the lifespan of normal human cells is not known. We investigated the possibility that critically short telomere length activates a DNA damage response pathway involving p53 and p21(WAF1) in aging cells. We show that the DNA binding and transcriptional activity of p53 protein increases with cell age in the absence of any marked increase in the level of p53 protein, and that p21(WAF1) promoter activity in senescent cells is dependent on both p53 and the transcriptional co-activator p300. Moreover, we detected increased specific activity of p53 protein in AT fibroblasts, which exhibit accelerated telomere loss and undergo premature senescence, compared with normal fibroblasts. We investigated the possibility that poly(ADP-ribose) polymerase is involved in the post-translational activation of p53 protein in aging cells. We show that p53 protein can associate with PARP and inhibition of PARP activity leads to abrogation of p21 and mdm2 expression in response to DNA damage. Moreover, inhibition of PARP activity leads to extension of cellular lifespan. In contrast, hyperoxia, an activator of PARP, is associated with accelerated telomere loss, activation of p53 and premature senescence. We propose that p53 is post-translationally activated not only in response to DNA damage but also in response to the critical shortening of telomeres that occurs during cellular aging.