Regulation of p53 activity by HIPK2: molecular mechanisms and therapeutical implications in human cancer cells.

The p53 protein is the most studied tumor suppressor and the p53 pathway has been shown to mediate cellular stress responses that are disrupted when cancer develops. After DNA damage, p53 is activated as transcription factor to directly induce the expression of target genes involved in cell-cycle arrest, DNA repair, senescence and, importantly, apoptosis. Post-translational modifications of p53 are essential for the activation of p53 and for selection of target genes. The tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) is a crucial regulator of p53 apoptotic function by phosphorylating its N-terminal serine 46 (Ser46) and facilitating Lys382 acetylation at the C-terminus. HIPK2 is activated by numerous genotoxic agents and can be deregulated in tumors by several conditions including hypoxia. Recent findings suggest that HIPK2 active/inactive protein can affect p53 function in multiple and unexpected ways. This makes p53 as well as HIPK2 interesting targets for cancer therapy. Hence, understanding the role of HIPK2 as p53 activator may provide important insights in the process of tumor progression, and may also serve as the crucial point in the diagnostic and therapeutical aspects of cancer.

Gene expression profiles of AML derived stem cells; similarity to hematopoietic stem cells.

Tumors contain a fraction of cancer stem cells that maintain the propagation of the disease. The CD34(+)CD38(-) cells, isolated from acute myeloid leukemia (AML), were shown to be enriched leukemic stem cells (LSC). We isolated the CD34(+)CD38(-) cell fraction from AML and compared their gene expression profiles to the CD34(+)CD38(+) cell fraction, using microarrays. We found 409 genes that were at least twofold over- or underexpressed between the two cell populations. These include underexpression of DNA repair, signal transduction and cell cycle genes, consistent with the relative quiescence of stem cells, and chromosomal aberrations and mutations of leukemic cells. Comparison of the LSC expression data to that of normal hematopoietic stem cells (HSC) revealed that 34% of the modulated genes are shared by both LSC and HSC, supporting the suggestion that the LSC originated within the HSC progenitors. We focused on the Notch pathway since Jagged-2, a Notch ligand was found to be overexpressed in the LSC samples. We show that DAPT, an inhibitor of gamma-secretase, a protease that is involved in Jagged and Notch signaling, inhibits LSC growth in colony formation assays. Identification of additional genes that regulate LSC self-renewal may provide new targets for therapy.

S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation.

The p53 paralogues p73, p63 and their respective truncated isoforms have been shown to be critical regulators of developmental and differentiation processes. Indeed, both p73- and p63-deficient mice exhibit severe developmental defects. Here, we show that S100A2 gene, whose transcript and protein are induced during keratinocyte differentiation of HaCaT cells, is a direct transcriptional target of p73beta and DeltaNp63alpha and is required for proper keratinocyte differentiation. Transactivation assays reveal that p73beta and DeltaNp63alpha exert opposite transcriptional effects on S100A2 gene. While DeltaNp63alpha is found in vivo onto S100A2 regulatory regions predominantly in proliferating cells, p73beta is recruited in differentiating cells. Silencing of p73 impairs the induction of S100A2 during the differentiation of HaCaT cells. Moreover, silencing of p73 or S100A2 impairs the proper expression of keratinocyte differentiation markers. Of note, p53 family members do not trigger S100A2 gene expression in response to apoptotic doses of cisplatin and doxorubicin.

The UVB-induced gene expression profile of human epidermis in vivo is different from that of cultured keratinocytes.

In order to obtain a comprehensive picture of the molecular events regulating cutaneous photodamage of intact human epidermis, suction blister roofs obtained after a single dose of in vivo ultraviolet (UV)B exposure were used for microarray profiling. We found a changed expression of 619 genes. Half of the UVB-regulated genes had returned to pre-exposure baseline levels at 72 h, underscoring the transient character of the molecular cutaneous UVB response. Of special interest was our finding that several of the central p53 target genes remained unaffected following UVB exposure in spite of p53 protein accumulation. We next compared the in vivo expression profiles of epidermal sheets to that of cultured human epidermal keratinocytes exposed to UVB in vitro. We found 1931 genes that differed in their expression profiles between the two groups. The expression profile in intact epidemis was geared mainly towards DNA repair, whereas cultured keratinocytes responded predominantly by activating genes associated with cell-cycle arrest and apoptosis. These differences in expression profiles might reflect differences between mature differentiating keratinocytes in the suprabasal epidermal layers versus exponentially proliferating keratinocytes in cell culture. Our findings show that extreme care should be taken when extrapolating from findings based on keratinocyte cultures to changes in intact epidermis.

Apoptosis of granulosa cells and female infertility in achondroplastic mice expressing mutant fibroblast growth factor receptor 3G374R.

Fibroblast growth factors play an important role in the control of ovarian folliculogenesis, but the complete repertoire of ovarian receptors which can transduce the fibroblast growth factor signals and their precise localization in the ovary have not yet been characterized. The most common form of inherited human dwarfism results from a point mutation in the transmembrane region of fibroblast growth factor receptor 3. A mouse model for achondroplasia was generated by introducing the human mutation (glycine 380-arginine) into the mouse fibroblast growth factor receptor 3 (G374R) by a "knock-in" approach using gene targeting leading to a constitutively active receptor. This resulted in the development of dwarf mice that share many features with human achondroplasia. Here we report that female (fibroblast growth factor receptor 3 G374R) dwarf mice become infertile. While no significant changes were observed in the anatomical and histological appearance of ovaries of 3-wk-old dwarf mice, a dramatic difference was observed in ovaries of 3-month-old mice. The normal ovary consists mainly of healthy corpora lutea and follicles at different stages of development, whereas the ovaries of the dwarf mice remain small and contain mainly follicles with a progressive apoptosis in the granulosa cells, and no corpora lutea could be observed. The levels of LH, FSH, and progesterone were lower by 72.3%, 38.0%, and 40.0%, respectively, in the blood of the dwarf mice compared with normal mice, and the total bioactivity of pituitary FSH and LH was lower by 65.6% and 79.6%, respectively, in the dwarf mice compared with normal mice. However treatment with PMSG and human CG of the dwarf mice led to rapid follicular development and formation of corpora lutea. Interestingly, the expression of the tumor suppressor gene p53 was increased dramatically in ovaries of the dwarf mice. The presence of the fibroblast growth factor receptor 3 cellular receptors in both normal and dwarf animals was demonstrated by Western blot and immunostaining. However, the distribution of the fibroblast growth factor receptors in the two strains shows significant differences. In the normal ovaries fibroblast growth factor receptor 3 was homogeneously distributed on the cell membrane of the granulosa cells and was absent in theca as well as corpora lutea cells, whereas in dwarf mice ovaries it was highly clustered on granulosa cells and very often appears in endocytic vesicles. Aged oocytes were more frequently observed in preantral follicles of ovaries of the dwarf mice. Nevertheless, oocytes isolated from antral follicles resume their meiotic division at a high percentage, similar to oocytes obtained from normal ovaries. The results imply fibroblast growth factor receptor 3 involvement in the control of follicular development through regulation of granulosa cell growth and differentiation, and that unovulation in the dwarf mice could be overcome in part by administration of exogenous gonadotropins. Moreover, it is suggested that the infertile phenotype is partially due to defects in the pituitary-gonadal axis.

DNA microarrays identification of primary and secondary target genes regulated by p53.

The transcriptional program regulated by the tumor suppressor p53 was analysed using oligonucleotide microarrays. A human lung cancer cell line that expresses the temperature sensitive murine p53 was utilized to quantitate mRNA levels of various genes at different time points after shifting the temperature to 32 degrees C. Inhibition of protein synthesis by cycloheximide (CHX) was used to distinguish between primary and secondary target genes regulated by p53. In the absence of CHX, 259 and 125 genes were up or down-regulated respectively; only 38 and 24 of these genes were up and down-regulated by p53 also in the presence of CHX and are considered primary targets in this cell line. Cluster analysis of these data using the super paramagnetic clustering (SPC) algorithm demonstrate that the primary genes can be distinguished as a single cluster among a large pool of p53 regulated genes. This procedure identified additional genes that co-cluster with the primary targets and can also be classified as such genes. In addition to cell cycle (e.g. p21, TGF-beta, Cyclin E) and apoptosis (e.g. Fas, Bak, IAP) related genes, the primary targets of p53 include genes involved in many aspects of cell function, including cell adhesion (e.g. Thymosin, Smoothelin), signaling (e.g. H-Ras, Diacylglycerol kinase), transcription (e.g. ATF3, LISCH7), neuronal growth (e.g. Ninjurin, NSCL2) and DNA repair (e.g. BTG2, DDB2). The results suggest that p53 activates concerted opposing signals and exerts its effect through a diverse network of transcriptional changes that collectively alter the cell phenotype in response to stress.

DNA microarray analysis of genes involved in p53 mediated apoptosis: activation of Apaf-1.

The transcription regulation activity of p53 controls cellular response to a variety of stress conditions, leading to growth arrest and apoptosis. Despite major progress in the understanding of the global effects of p53 on cellular function the pathways by which p53 activates apoptosis are not well defined. To study genes activated in the p53 induced apoptotic process, we used a mouse myeloid leukemic cell line (LTR6) expressing the temperature-sensitive p53 (val135) that undergoes apoptosis upon shifting the temperature to 32 degrees C. We analysed the gene expression profile at different time points after p53 activation using oligonucleotide microarray capable of detecting approximately 11,000 mRNA species. Cluster analysis of the p53-regulated genes indicate a pattern of early and late induced sets of genes. We show that 91 and 44 genes were substantially up and down regulated, respectively, by p53. Functional classification of these genes reveals that they are involved in many aspects of cell function, in addition to growth arrest and apoptosis. Comparison of p53 regulated gene expression profile in LTR6 cells to that of a human lung cancer cell line (H1299) that undergoes growth arrest but not apoptosis demonstrates that only 15% of the genes are common to both systems. This observation supports the presence of two distinct transcriptional programs in response to p53 signaling, one leading to growth arrest and the other to apoptosis. The proapoptotic genes induced only in LTR6 cells like Apaf-1, Sumo-1 and gelsolin among others may suggest a possible explanation for apoptosis in LTR6 cells.

FGF receptor mutations: dimerization syndromes, cell growth suppression, and animal models.

This review describes recent progress in the field of fibroblast growth factor receptors (FGFRs) with an emphasis on the role of FGFR mutants in skeletal malformations. This family of four receptors contains the most frequent germline mutations in humans. More than 75 mutations have been recorded, which account for more than seven skeletal syndromes. The common cause for all the mutant phenotypes is gain-of-function by receptor activation through three major mechanisms: receptor dimerization, kinase activation, and increased affinity for FGF. The severity of the disease is correlated with both the extent of receptor activation and the specific tissue in which the mutant receptor form is expressed. Paradoxically, the consequence of receptor activation is inhibition of chondrocyte cell growth through signaling pathways that are cell-type specific. The structure of the FGFR-FGF complex and its possible ternary complex with heparin explain the mechanism of receptor dimerization in the ectodomain and the possible contribution by some of the mutations to this process. Analysis of FGFR3 mutant mice produced by gene targeting as models for human disease, and studies in cell lines, have begun to delineate the novel signaling pathways of FGFR3 and to define possible targets for therapy.

Profile of gene expression regulated by induced p53: connection to the TGF-beta family.

The transcription regulatory function of p53 was analyzed by using two inducible p53 systems in the human lung cancer cell line H1299. cDNA probes derived from RNA harvested 12 h after p53 induction were used to probe filters containing cDNA arrays. Over 20 genes were found to be significantly induced or suppressed by p53. The induced genes can be classified mainly as cell cycle inhibitors like p21waf, GADD45, apoptosis-related genes like Fas/APO1 and PIG3 or DNA repair genes like DDB2, DNA ligase and G/T mismatch DNA glycosylase. The suppressed genes include mainly cell cycle regulators like cyclin B1, cyclin H and kinases like c-abl, CLK1 and others. The most notable induced gene was MIC-1, encoding a TGF-beta-related secretory protein, suggesting a potential paracrine component for p53 growth suppression.

Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice.

Achondroplasia, the most common genetic form of human dwarfism, results from a point mutation (G380R) in the gene for fibroblast growth factor receptor 3 (FGFR-3). Heterozygotes for the mutation share disproportionate, proximal shortening of the limbs, mid-face hypoplasia and relative macrocephaly due to a failure in endochondral ossification. Here we have generated transgenic mice expressing the human mutant FGFR-3 under the transcriptional control of the mouse gene. Mice that are hemizygous for the mutant human gene display disproportionate dwarfism with skeletal phenotypes remarkably similar to those of human achondroplasia. Mice that are homozygous for the transgene suffer from a profound delay in skeletal development and die at birth, similar in that respect to humans homozygous for the achondroplasia mutant gene. Microscopic analysis of long bones demonstrates growth plate morphology compatible with that of human achondroplasia cases, sharing endochondral growth inhibition with restrained chondrocyte proliferation and maturation, penetration of ossification tufts and aberrant vascularization.

Induced p21waf expression in H1299 cell line promotes cell senescence and protects against cytotoxic effect of radiation and doxorubicin.

The CDK inhibitor p21waf is a principal mediator of p53 function but can also be transactivated by many p53-independent stimuli leading to cell growth arrest or differentiation. In order to study the function of p21waf in a p53-deficient environment, we established an inducible expression of p21waf in the p53-null lung cancer cell line H1299, based on the muristerone-regulated system. Overexpression of p21waf led cells to growth arrest which after several days became irreversible and the arrested cells acquired a senescent phenotype as judged by cell shape, the senescence-associated beta-gal marker and inhibition of colony formation. The effect of p21waf overexpression, in the absence of p53, on the cytotoxicity caused by irradiation, doxorubicin and taxol was studied. Expression of p21waf provided protection against the cytotoxic effect of radiation and doxorubicin but not of taxol. These results are relevant to treatment of cancer when p53 is inactive.

H2O2-induced apoptotic death in serum-deprived cultures of oligodendroglia origin is linked to cell differentiation.

When deprived of serum, oligodendroglialike (OLN 93) cells grown on poly-L-lysine-coated culture dishes cease to proliferate after 3 days and morphologically extend many fibers resembling morphologically differentiated, immature oligodendrocytes. At this time no cell death is apparent unless serum deprivation is extended for a period longer than 1 week. After 3 days in serum-deprived medium, treatment of cells with 1 mM H2O2 for 30 min facilitates apoptotic cell death, even when serum is added during the recovery period. Both serum-deprived, differentiated cells, and proliferating cells, respond to H2O2 by an initial growth arrest followed by growth resumption after 48 hr. However proliferating cells show resistance to the apoptotic effect of H2O2. This is correlated with growth arrest in the S phase at different stages of DNA replication, as well as with different timing of induced p21Waf1 expression. Thus, cells grown in serum, express elevated p21Waf1 protein levels after 4 hr, whereas serum-deprived, differentiated cells, only after 24 hr. The mRNA levels of p21Waf1 follow a similar timed pattern. Hence p21Waf1 may protect OLN 93 cells against the genotoxic effect of H2O2. The data suggest an intimate relationship between G1-arrest, morphological differentiation, and H2O2-mediated apoptosis.

A mouse model for achondroplasia produced by targeting fibroblast growth factor receptor 3.

Achondroplasia, the most common form of dwarfism in man, is a dominant genetic disorder caused by a point mutation (G380R) in the transmembrane region of fibroblast growth factor receptor 3 (FGFR3). We used gene targeting to introduce the human achondroplasia mutation into the murine FGFR3 gene. Heterozygotes for this point mutation that carried the neo cassette were normal whereas neo+ homozygotes had a phenotype similar to FGFR3-deficient mice, exhibiting bone overgrowth. This was because of interference with mRNA processing in the presence of the neo cassette. Removal of the neo selection marker by Cre/loxP recombination yielded a dominant dwarf phenotype. These mice are distinguished by their small size, shortened craniofacial area, hypoplasia of the midface with protruding incisors, distorted brain case with anteriorly shifted foramen magnum, kyphosis, and narrowed and distorted growth plates in the long bones, vertebrae, and ribs. These experiments demonstrate that achondroplasia results from a gain-of-FGFR3-function leading to inhibition of chondrocyte proliferation. These achondroplastic dwarf mice represent a reliable and useful model for developing drugs for potential treatment of the human disease.

Induced p53 expression in lung cancer cell line promotes cell senescence and differentially modifies the cytotoxicity of anti-cancer drugs.

The p53-null human lung cancer cell line H1299 was used in order to generate clones with ecdysone-inducible p53 as well as ecdysone-inducible p21waf1. Induced expression of p53 resulted in irreversible cell growth arrest with characteristics of replicative senescence, suggesting that p53 can prevent immortalization by activating a senescence program. The effect of induced p53 and p21waf1 expression on the cytotoxic action of the anti-cancer drugs etoposide and cisplatin was also analysed. Whereas p21waf1 overexpression conferred increased resistance to killing by either drug, p53 overexpression enhanced the cytotoxic effect of cisplatin but protected against etoposide cytotoxicity. These results imply that the impact of p53 on susceptibility to chemotherapy may depend greatly on the particular drug and type of DNA damage. Moreover, these data demonstrate the importance of using isogenic cell lines to address this issue.

Overexpression of p21waf1/cip1 arrests the growth of chicken embryo fibroblasts that overexpress E2F1.

P21waf1/cip1 is a potent inhibitor of cell cycle progression and can inhibit the growth of both normal cells and cells transformed by a number of oncogenes. However, the ability of p21waf1/cip1 to inhibit the growth of cells that overexpress the transcriptional transactivator E2F1 is controversial: it has been reported both that E2F1 can and cannot overcome the block in the cell cycle induced by p21waf1/cip1. To avoid the complications that arise when such experiments are done with permanent cell lines, we tested the effects of overexpressing p21waf/cip1 and E2F1 in primary chicken embryo fibroblasts. In this system very high levels of E2F1 overexpression cause considerable apoptosis; however, the surviving cells still overexpress E2F1. These cells are transformed and their growth is blocked by overexpression of p21waf1/cip1.

Asparagine-344 is a key residue for ligand binding in keratinocyte growth factor receptor.

The membrane proximal, immunoglobulin- (Ig-) like domain 3 of KGFR shows significant sequence similarity to the Ig light chain variable (V) domain. According to our model, based on this similarity, the F-G loop in KGFR corresponds to the complementarity determining region (CDR) 3 of the Ig V domain. The F-G loop in the membrane proximal domain of the keratinocyte growth factor receptor has previously been shown to participate in determining the FGF ligand binding specificity of KGFR [Gray, T. E., Eisenstein, M., Shimon, T., Givol, D., & Yayon, A. (1995) Biochemistry 34, 10325-10333]. Here, we report the effects of additional mutations in this F-G loop. Both a single mutant KGFR Q348-->I and a double mutant KGFR Q348-->I, Q351-->H are found to have relatively mild effects on ligand binding, as was previously found for three other F-G loop mutant receptors. In contrast, a single mutation N344-->A in the F-G loop of KGFR is sufficient to abolish essentially all affinity of this receptor for its primary ligand KGF, while some affinity for aFGF is retained. Asparagine-344 is, therefore, essential for ligand binding by KGFR. We discuss the likelihood of this effect being due to global or local structural changes or to the removal of a specific interaction with the ligand, in relation to various known and model structures. Taking into account the mild effects of other mutations in the region and various other considerations, we tend to favor the idea that asparagine-344 is a key residue in determining the local conformation of the F-G loop.

Inhibition of oncogene-mediated transformation by ectopic expression of p21Waf1 in NIH3T3 cells.

The p53-regulated p21Waf1 protein is a universal inhibitor of cyclin-dependent kinases (CDKs). To study the potential tumor-suppressive properties of CDK inhibitors, the ability of p21Waf1 to interfere with oncogene-mediated cellular transformation was analysed in the NIH3T3 cell system. Cotransfection of waf1 together with activated ras or several other oncogenes into NIH3T3 cells potently inhibited the formation of transformed foci in a dose-dependent manner. Expression of the CDK-binding N-terminal half of p21Waf1 (N-p21Waf1) was necessary and sufficient to inhibit Ras-induced focus formation. In contrast, expression of the C-terminal domain (C-p21Waf1) had no effect on Ras-induced focus formation. Immunofluorescence analysis revealed that ectopically expressed p21Waf1 and C-p21Waf1 were localized in the nucleus, while N-p21Waf1 was found in the cytoplasm, with the tendency to accumulate around the nuclear membrane. Surprisingly, stable NIH3T3 transfectants expressing ectopic p21Waf1 grew at the same rate and displayed similar cell cycle distribution as NIH3T3 cells transfected with the same vector containing no insert. However, ectopic p21Waf1 expression did inhibit Ras-mediated anchorage-independent colony formation, indicating that p21Waf1 can selectively interfere with oncogene-mediated transformation without affecting NIH3T3 cell growth, at least at the levels of p21Waf1 expression achieved in these experiments. Transient transfection of waf1 into NIH3T3 cells inhibited Ras-induced transcription from a E2F-responsive element but not from a serum-responsive element, indicating that p21Waf1 acts downstream of early transcriptional events induced by Ras but upstream of E2F-controlled gene transcription. These results provide evidence that p21Waf1 potently suppresses oncogene-mediated cellular transformation of NIH3T3 cells and that it may do so by inhibiting E2F-driven transcription of S phase genes.

Overexpression of human p21waf1/cip1 arrests the growth of chicken embryo fibroblasts transformed by individual oncogenes.

In normal cells, cell growth and division is controlled by the interplay between proto-oncogenes and tumor suppressor genes. Cancer cells usually have both activated an oncogene and have lost a functional tumor suppressor gene. High level expression of a tumor suppressor, p53, can block the growth of cancer cells. waf1/cip1 is transactivated by the tumor suppressor p53 and the p21waf1/cip1 protein is itself a suppressor of cell growth. To test the effect of growth suppression genes on the growth of cells transformed by individual oncogenes, we have used replication-competent retroviral vectors to induce high level expression of p53 and p21waf1/cip1. Overexpression of p21waf1/cip1 arrests the growth of chicken embryo fibroblasts (CEF) transformed by v-Src, tf-Ras, c-Mos and c-Myc. These data suggest that p21waf1/cip1 might be a useful tool in gene therapy for human cancer.

Molecular modeling based mutagenesis defines ligand binding and specificity determining regions of fibroblast growth factor receptors.

The fibroblast growth factor receptor 2 (FGFR2) and the keratinocyte growth factor receptor (KGFR) have different ligand binding specificities despite differing only in the second half of their immunoglobulin-like (Ig-like) domain III. Three-dimensional model structures were generated for domain III on the basis of variable (V) Ig domains. The region that differs between the two receptors is predicted to include two loops: one connects beta-strands F-G and is analogous to the complementarity determining region 3 (CDR3) of immunoglobulins; the other connects beta-strands D-E. These regions were targeted for mutagenesis. Single mutations in the F-G loop were found to only slightly alter ligand binding, whereas a double mutant, KGFR Y345-->S,Q348-->I, acquired significant affinity for bFGF. Notably, the affinity of this double mutant KGFR for KGF and aFGF was essentially unaltered. A mutant FGFR2, in which the D-E beta-hairpin (T319TDKEI) is replaced with the KGFR D-E beta-hairpin (S319SNA), has 9-fold reduced affinity for bFGF. These results demonstrate that the F-G or CDR3 analogous loop in FGFRs plays a key role in determining ligand binding and specificity. In addition, however, the protein loop connecting beta-strands D and E may also be involved in ligand binding. Several point mutations in FGFR2, shown recently to give rise to multiple inherited skeletal defects, are localized according to our models to the F-G or D-E loops of domain III. Our results strongly suggest that these naturally occurring mutations specifically alter ligand binding by FGFR2.

The tumor suppression function of p21Waf is contained in its N-terminal half ('half-WAF').

The Waf-1 encoded protein, p21, mediates p53 suppression of tumor cell growth. Overexpression of p21 in the H1299 tumor cell line suppresses colony formation similar to that resulted from p53 overexpression. In an effort to localize the tumor suppression function within the structure of p21 we utilized vectors constructed with systematic truncations of p21 and tested their efficiency in suppressing tumor cell growth. We demonstrate that the N-terminal half of the p21 molecule (residues 1-80 and 1-89) shows better tumor cell growth suppression than the entire p21 molecule whereas the C-terminal half of p21 does not show this effect. These results may have implications for gene therapy of cancer.