The effects of IL -10 producing monocytes on T-cell activation in patients with epithelial ovarian cancer

A newly described subset of monocytes has been identified in peritoneal exudate cells (PEC) from the malignant ascites of patients with ovarian cancer. These cells were characterized by the production of IL-10 and TGF-β2, but not IL-12, IL-1α, or TNF-α, and expressed CD14, CD16, and CD54, but not HLA-DR, CD80, CD86, CD11a, CD11b, or CD25 cell surface antigens. Since this subset of monocytes could affect the modulation of tumor immune responses in vivo, studies were undertaken to determine their effect on the activation and proliferation of autologous T-cells from the peritoneal cavity of patients with ovarian carcinoma. Cytokine transcripts, including IL-2, GM-CSF, and IFN-γ were detected in T-cells isolated from patient specimens that also contained the IL-10 producing monocytes, although the IFN-γ and IL-2 proteins could not be detected in T-cells co-incubated with the IL-10 producing monocytes in vitro. Additionally, IL-10 producing monocytes co-cultured with autologous T-cells inhibited the proliferation of the T-cells in response to PHA. T-cell proliferation and cytokine protein production could be restored by the addition of neutralizing antibodies to IL-10R and TGF-β to the co-culture system. These results suggested that this subset of monocytes may modulate antitumor immune responses by inhibiting T-cell proliferation and cytokine protein production. Further studies determined that the precursors to the inhibitory monocytes were tumor-associated and only present in the peripheral blood of patients with ovarian cancer and not present in the peripheral blood of healthy donors. These precursors could be induced to the suppressor phenotype by the addition of IL-2 and GM-CSF, two cytokines detected in the peritoneal cavity of ovarian cancer patients. Lastly, it was shown that the suppressor monocytes from the peritoneal cavity of ovarian cancer patients could be differentiated to a non-inhibitory phenotype by the addition of TNF-α and IFN-γ to the culture system. The differentiated monocytes did not produce IL-10, expressed the activation antigens HLA-DR, CD80, and CD86, and were able to stimulate autologous T-cells in vitro. Since a concomitant reduction in immune function is associated with tumor growth and progression, the effects of these monocytes are of considerable importance in the context of tumor immunotherapy. ^

A novel DNA damage inducible inhibitor of p53

p53 is required for the maintenance of the genomic stability of cells. Mutations in the p53 tumor-suppressor gene occur in more than 50% of human cancers of diverse types. In addition, 70% of families with Li-Fraumeni syndrome have a germline mutation in p53, predisposing these individuals to multiple forms of cancer. In response to DNA damage, p53 becomes stabilized and activated. However the exact mechanism by which DNA damage signals the stabilization and activation of p53 still remains elusive. The biochemical activity of p53 that is required for tumor suppression, and presumably the cellular response to DNA damage, involves the ability of the protein to bind to specific DNA sequences and to function as a transcription factor. For the downstream targets, p53 transactivates many genes involved in growth arrest, apoptosis and DNA repair such as p21, Bax and GADD45, respectively. An open question in the field is how cells can determine the downstream effects of p53. ^ We hypothesize that, through its associated proteins, p53 can differentially transactivate its target genes, which determine its downstream effect. Additionally, p53 interacting proteins may be involved in signaling for the stabilization and activation of p53. Therefore, a key aspect to understanding p53 function is the identification and analysis of proteins that interact with it. We have employed the Sos recruitment system (SRS), a cytoplasmic yeast two-hybrid screen to identify p53 interacting proteins. The SRS is based on the ability of Sos to activate Ras when it becomes localized to the plasma membrane. The system takes advantage of an S. cerevisiae strain, cdc25-2 temperature sensitive mutant, harboring a mutation in Sos. In this strain, fusion proteins containing a truncated Sos will only localize to the membrane by protein-protein interaction, which allows growth at non-permissive temperature. This system allows the use of intact transcriptional activators such as p53. ^ To date, using a modified SRS library screen to identify p53 interacting proteins, I have identified p53 (known to interact with itself) and a novel p53-interacting protein (PIP). PIP is a specific p53 interacting protein in the SRS. The interaction of p53 and PIP was further confirmed by performing in vitro and in vivo binding assays. In the in vivo binding study, the interaction can only be detected in the presence of ionizing radiation suggesting that this interaction might be involved in DNA-damage induced p53-signalling pathway. After screening cDNA and genomic libraries, a full-length PIP-cDNA clone ( ∼ 3kb) was obtained which encodes a protein of 429 amino acids with calculated molecular weight of 46 kDa. The results of genebank search indicated that the PIP is an unidentified gene and contains a conserved ring-finger domain, which is present in a diverse family of regulatory proteins involved in different aspects of cellular function. Northern blot analysis revealed that the size of its messenge is approximately 3 kb preferentially expressed in brain, heart, liver and kidney. The PIP protein is mainly located in the cytoplasm as determined by the cellular localization of a green fluorescence fusion protein. Preliminary functional analysis revealed that PIP downregulated the transactivation activity of p53 on both p21 and mdm2 promoters. Thus, PIP may be a novel negative regulator of p53 subsequent to DNA damage. ^

The role of p53 in skin cancer induction by UV radiation and as a potential tumor antigen in UV-induced murine skin tumors

Carcinoma of the skin is the most common type of human cancer in the United States. Ultraviolet radiation (UVR) present in the sunlight is thought to be the major carcinogen responsible for induction of skin cancer. In UV-associated skin carcinogenesis, mutations in p53 are not only present with very high frequency, but occur early in the course of tumor development. In addition, UV-induced skin tumors in mice exhibit unique immunological characteristics. They are highly antigenic and express both individually-specific tumor transplantation antigens recognized by effector T cells and the UV-associated common antigen recognized by UV-induced suppressor T cells. ^ To examine the hypothesis that p53 plays a critical role in preventing skin cancer induction by UVR, mice constitutively lacking one or two functional p53 alleles were compared to wild-type mice for their susceptibility to UV carcinogenesis. Both p53 +/– and –/– mice showed greater susceptibility to skin cancer induction than wild-type mice, and –/– mice were the most susceptible, Accelerated tumor development in the p53 +/– mice was not associated with loss of the remaining wild-type allele of p53 , but in many cases was associated with UV-induced mutations in p53. Our studies clearly demonstrate the essential role of p53 in protection against UV carcinogenesis, particularly in the eye and epidermis. ^ The role of p53 in the antigenicity of UV-induced murine skin tumors was also addressed. Primary UV-induced tumors from p53 –/–, +/– and +/+ mice were transplanted into both normal and immunosuppressed mice, and rates of tumor rejection were compared. Tumors from mice with only one or no functional p53 alleles were less antigenic than those from mice with two functional p53 alleles. Moreover, tumors with no functional p53 also failed to grow well in chronically UV-irradiated mice. These results indicate that p53 contributes to the strong antigenicity of UV-induced murine skin tumors, and suggest that it may play a critical role in expression of the UV-associated common antigen recognized by suppressor T cells. ^ In this study we also monitored the effect of UVR on the development of lymphoid malignancies in p53 deficient mice. The incidence of lymphoid malignancies in UV-irradiated p53 +/– mice was drastically enhanced compared to that in unirradiated counterparts. The immune responses of the mice were identical and were suppressed to the same extent by UV irradiation regardless of the p53 genotype. These data provide the first experimental evidence that exposure to UVR can contribute to the development of lymphoid neoplasms in genetically susceptible hosts. ^

Dual role of interleukin-12 on ultraviolet -induced immune suppression

Skin cancer is the most prevalent form of neoplasia, with over one million newcases diagnosed this year. UV radiation is a ubiquitous environmental agent that induces skin cancer. In addition to its carcinogenic effect, UV radiation also suppresses cell-mediated immune responses. This immune suppression is not only observed at the site of irradiation, but UV radiation also induces systemic immune suppression. Since UV radiation has a limited ability to penetrate the skin, the question of the mechanism of this systemic immune suppression arises. A number of studies have suggested that UV radiation induce systemic effects through the production of immunoregulatory cytokines, such as IL-4 and IL-10. These cytokines affect the immune response by altering systemic antigen presentation, specifically by suppressing the activation of Th1 cells while allowing the activation of Th2 cells. Because IL-12 is an important regulator of Th1 cell activation, we tested the hypothesis that administration of IL-12 could overcome UV-induced immune suppression. ^ The studies presented here are divided into dime specific aims. In the first specific aim, the ability of IL-12 to overcome UV-induced immune suppression was examined. IL-12 could overcome UV-induced immune suppression as well as prevent the generation of and neutralize the activity of preformed suppressor cells induced by UV radiation. In the second specific aim, the mechanism by which IL-12 overcomes UV-induced immune suppression was examined. IL-12 overcame UV-induced immune suppression by blocking the production of immunoregulatory cytokines such as IL-4, IL-10 and TNF-α. In the third specific aim, the effect of UV radiation on antigen presentation was investigated. UV radiation was found to decrease the production of biologically active IL-12. In addition, UV also increased the production of IL-12p40 homodimer, an antagonist of IL-12p70 heterodimer. This result suggests that IL-12 may have a dual role in the immune suppression induced by, UV radiation. On one hand the biologically active IL-12p70 heterodimer blocks UV-induced immune suppression. In contrast, IL-12p40 homodimer may mediate the suppressive effect of UV radiation. This paradox indicates that IL-12 may have a greater regulatory role in the immune response than was previously suspected. ^

Functional activation of p53 induces apoptosis in the absence of MDM2

The p53 tumor suppressor gene product is negatively regulated by the product of its downstream target, mdm2. The mdm2 oncogene abrogates p53 transactivation function. Amplification of mdm2 occurs in 36% of human sarcomas, which often retain p53 in wild type form, suggesting that overexpression of mdm2 in tumors results in p53 inactivation. Thus, the relationship of p53 to mdm2 is important in tumorigenesis. The deletion of mdm2 in the mouse results in embryonic lethality by 5.5 days post coitum. Embryonic lethality of the mdm2 null embryos was overcome by simultaneous loss of the p53 tumor suppressor, which substantiates the importance of the negative regulatory function of MDM2 on p53 function in vivo. These data suggest that the loss of MDM2 function allowed the constitutively active p53 protein to induce either a complete G1 arrest or the p53-dependent apoptotic pathway, resulting in the death of the mdm2−/− embryos.^ The present study examines the hypothesis that the absence of mdm2 induces apoptosis due to p53 activation. Viability of the p53−/−mdm2−/− mice has allowed establishment of mouse embryo fibroblasts (MEFs) and a detailed examination of the properties of these cells. To introduce p53 into this system, and essentially recreate a mdm2 null cell, a temperature sensitive p53 (tsp53) point mutant (A135V) was used, which exhibits a nonfunctional, mutant conformation at 39°C and wild type, functional conformation at 32°C. Infected pools of p53−/− and p53−/−mdm2−/− MEFs with the tsp53 gene were established and single-cell clonal populations expressing tsp53 were selected. Shifting the cells from 39°C to 32°C caused p53−/−mdm2 −/− lines expressing tsp53 to undergo up to 80% apoptosis, which did not occur in the p53−/− lines expressing tsp53 nor the parental lines lacking p53 expression. Furthermore, the amount of p53 present in the clonal population determined the extent of apoptosis. Tsp53 is transcriptionally active in this system, however, it discriminates among different target promoters and does not induce the apoptosis effector targets bax or Fas/Apo1. ^ In summary, this study indicates that the presence or absence of mdm2 is the determining factor for the ability of p53 to trigger apoptosis in this system. The loss of mdm2 promotes p53-dependent apoptosis in MEFs in a cell cycle and dose-dependent manner. p53 is differentially phosphorylated in the presence and absence of mdm2, but does not induce the apoptosis effectors, bax or Fas/ Apo1. ^

Participation of bcl -2 and bax in epidermal homeostasis and non-melanoma skin cancer

Non-melanoma skin cancer (NMSC) is the most frequently diagnosed form of cancer in United States. As in many other cancers, this slow growing malignancy manifests deregulated expression of apoptosis regulating proteins including bcl-2 family member proteins. To understand the role of apoptosis regulating protein in epidermal homeostasis and progression of NMSC, we investigated keratinocyte proliferation, differentiation and tumorigenesis in bcl-2 and bax null mice. The rate and the pattern of proliferation and spontaneous cell death were the same between the null and the control mice. Both bcl-2 and bax null epidermis showed decreased levels of cytokeratin 14 expression compared to the control littermates. Also, the gene knock out mice showed higher expression of cytokeratin 1 and loricrin in epidermis compared to the control mice. The apoptotic response to genotoxic agent, UV radiation (UVR), was assessed by counting sunburn cells. The bax null keratinocytes showed a resistance to apoptosis while bcl-2 null mice showed an increased susceptibility to cell death compared to the control mice. Moreover, we demonstrated an increase in tumor incidence in bax null mice compared to control littermates in the in vivo chemical carcinogenesis study. Next, we examined the tumor suppressor role of bax protein in NMSC by studying its participation in repair of UVR-mediated DNA lesions. In UVR treated primary keratinocytes from bax deficient mice, the level of CPD remaining was twice that of control cells at 48 hours. Similar results were obtained using embryonic fibroblasts from bax null and bax +/+ embryos, and also with a bax deficient prostate cancer cell line in which bax expression had been restored. However, the repair rate of 6-4 PP was unaffected by the absence of bax protein in all three of above mentioned cell types. In conclusion, bax protein may have a dual function in its role as tumor suppressor in NMSC. Bax may directly or indirectly facilitate DNA repair, or programmed cell death if DNA damage is too severe, thus, in either function, preserving genomic integrity following a genotoxic event. ^

Rescue of embryonic lethality in mdmx null mice by loss of p53 suggests a non-overlapping pathway with MDM2 to regulate p53

The tumor suppressor p53 is mutated in over 50% of human sporadic tumors originating from diverse tissues. p53 responds to DNA damage and cell stress by activating the transcription of a variety of target genes, the protein products of which then initiate either growth arrest or apoptosis. ^ A p53 target with a particularly intriguing function is the oncogene MDM2. MDM2 functions, in part, by binding to and inhibiting p53's activity. Overexpression of MDM2, by gene amplification, has been found in 30% of human sarcomas harboring a wild type p53, indicating that an increase in MDM2 levels is sufficient for p53 inactivation. Mice carrying a homozygous null allele for mdm2 exhibit an early embryonic lethality that is completely rescued in a p53-null background. These data indicate that MDM2's only critical function in early mouse embryogenesis is the negative regulation of p53. ^ The mdmx gene is the first additional member of the mdm2 gene family to be isolated. MDMX, like MDM2, contains a RING-finger domain, ATP binding domain and a p53 binding domain, which retains the ability to bind and inhibit p53 transactivation in vitro. However, mdmx does not appear to be transcriptionally regulated by p53. We have cloned and characterized the murine mdmx genomic locus from a mouse 129 genomic library. The mdmx gene contains 11 exons, spans approximately 37 Kb of DNA, and is located on mouse chromosome 1. The genomic organization of the mdmx gene is identical to that of mdm2 except at the 5′ end of the gene near the p53 responsive element. Northern expression analysis of mdmx transcripts during mouse embryogenesis and in adult tissues revealed constitutive and ubiquitous expression throughout adult tissues and embryonic development. To determine the in vivo function of MDMX, mice carrying a null allele of mdmx have been generated. Mdmx homozygous null mice are early embryonic lethal. Mdmx null mice do not develop beyond 9.5 dpc and can be discerned by gross dissection as early as 7.5 dpc. Utilizing TUNEL and BrdU assays on 7.5 dpc histological sections we have determined that the mutant embryos are dying due to increased levels of growth arrest, but not apoptosis. Surprisingly, Mdmx homozygous null mice are viable in a p53 null background, indicating that MDMX is also very important in the negative regulation of p53. ^

The role of Ski/Sno oncoproteins as negative regulators of the TGF-beta/SMAD signaling pathway in B -cell non -Hodgkin's lymphoma (NHL-B)

Non-Hodgkin's lymphomas are common tumors of the human immune system, primarily of B cell lineage (NHL-B). Negative growth regulation in the B cell lineage is mediated primarily through the TGF-β/SMAD signaling pathway that regulates a variety of tumor suppressor genes. Ski was originally identified as a transforming oncoprotein, whereas SnoN is an isoform of the Sno protein that shares a large region of homology with Ski. In this study, we show that Ski/SnoN are endogenously over-expressed both in patients' lymphoma cells and NHL-B cell lines. Exogenous TGF-β1 treatment induces down-regulation of Ski and SnoN oncoprotein expression in an NHL-B cell line, implying that Ski and SnoN modulate the TGF-β signaling pathway and are involved in cell growth regulation. Furthermore, we have developed an NHL-B cell line (DB) that has a null mutation in TGF-β receptor type II. In this mutant cell line, Ski/SnoN proteins are not down-regulated in response to TGF-β1 treatment, suggesting that downregulation of Ski and SnoN proteins in NHL-B require an intact functional TGF-β signaling pathway Resting normal B cells do not express Ski until activated by antigens and exogenous cytokines, whereas a low level of SnoN is also present in peripheral blood Go B cells. In contrast, autonomously growing NHL-B cells over-express Ski and SnoN, implying that Ski and SnoN are important cell cycle regulators. To further investigate a possible link between reduction of the Ski protein level and growth inhibition, Ski antisense oligodeoxynucleotides were transfected into NHL-B cells. The Ski protein level was found to decrease to less than 40%, resulting in restoring the effect of TGF-β and leading to cell growth inhibition and G1 cell cycle arrest. Co-immunoprecipitation experiments demonstrated that Ski associates with Smad4 in the nucleus, strongly suggesting that over-expression of the nuclear protein Ski and/or SnoN negatively regulates the TGF-β pathway, possibly by modulating Smad-mediated tumor suppressor gene expression. Together, in NHL-B, the TGF-β/SMAD growth inhibitory pathway is usually intact, but over-expression of the Ski and/or SnoN, which binds to Smad4, abrogates the negative regulatory effects of TGF-β/SMAD in lymphoma cell growth and potentiates the growth potential of neoplastic B cells. ^

Structural and functional analysis of p84N5

Normal development and tissue homeostasis requires the carefully orchestrated balance between cell proliferation and cell death. Cell cycle checkpoints control the extent of cell proliferation. Cell death is coordinated through the activation of a cell suicide pathway that results in the morphologically recognizable form of death, apoptosis. Tumorigenesis requires that the balance between these two pathways be disrupted. The tumor suppressor protein Rb has not only been shown to be involved in the enforcement of cell cycle checkpoints, but has also been implicated in playing a role in the regulation of apoptosis. The manner in which Rb enforces cell cycle checkpoints has been well studied; however, its involvement in the regulation of apoptosis is still very unclear. p84N5 is a novel nuclear death domain containing protein that has been shown to interact with the N-terminus of Rb. The fact that it contains a death domain and the fact that it is nuclear localized possibly provides the first known mechanism for apoptotic signaling from the nucleus. The following study tested the hypothesis that the novel exclusively nuclear death domain containing protein p84N5 is an important mediator of programmed cell death and that its apoptotic function is reliant upon its nuclear localization and is regulated by unique functional domains within the p84N5 protein. We identified the p84N5 nuclear localization signal (NLS), eliminated it, and tested the functional significance of nuclear localization by using wild type and mutant sequences fused to EGFP-C1 (Clontech) to create wild type GFPN5 and subsequent mutants. The results of these assays demonstrated exclusive nuclear localization of GFPN5 is required for normal p84N5 induced apoptosis. We further conducted large-scale mutagenesis of the GFPN5 construct to identify a minimal region within p84N5 capable of interacting with Rb. We were able to identify a minimal sequence containing p84N5 amino acids 318 to 464 that was capable of interacting with Rb in co-immunoprecipitation assays. We continued by conducting a structural and functional analysis to identify the region or regions within p84N5 responsible for inducing apoptosis. Point mutations and small-scale deletions within the death domain of p84N5 lessened the effect but did not eliminate p84N5-induced cytotoxicity. Further analysis revealed that the minimal sequence of 318 to 464 of p84N5 was capable of inducing apoptosis to a similar degree as wild-type GFPN5 protein. Since amino acids 318 to 464 of p84N5 are capable of inducing apoptosis and interacting with Rb, we propose possible mechanisms whereby p84N5 may function in a Rb regulated manner. These results demonstrate that p84N5 induced apoptosis is reliant upon its nuclear localization and is regulated by unique functional domains within the p84N5 protein. ^

Genome instability quantified in constitutive tissues as well as putatively stable tumor tissue in hereditary non -polyposis colon cancer patients by small pool PCR

Microsatellite instability (MSI) is a hallmark of the mutator phenotype associated with Hereditary Non-Polyposis Colon Cancer (HNPCC). The MSI-High (MSI-H) HNPCC population has been well characterized, but the microsatellite low and stable (MSI-L/MSS) HNPCC population is much less understood. We hypothesize there are significant levels of MSI in HNPCC DNA classified as MSI-L/MSS, but no single variant allele makes up a sufficient population in the tumor DNA to be detected by standard analysis. Finding variants would suggest there is a mutator phenotype for the MSI-L/MSS HNPCC population that is distinct from the MSI-H HNPCC populations. This study quantified and compared MSI in HNPCC patients previously shown to be MSI-H, MSI-L/MSS and an MSI-H older, sporadic colorectal cancer patient. Small-pool Polymerase Chain Reactions (SP-PCRs) were conducted where the DNAs from each sample and controls are diluted into multiple pools, each containing approximately single genome equivalents. At least 100 alleles/sample were studied at six microsatellite loci. Mutant fragments were identified, quantified, and compared using Poisson statistics. Most of the variants were small deletions or insertions, with more mutants being deletions, as has been previously described in yeast and transgenic mice. SP-PCR, where most of the pools contained only 3 or less fragments, enabled identification of variants too infrequent to be detected by large pool PCR. Mutant fragments in positive control MSI-H tumor samples ranged from 0.26 to 0.68 in at least 4 of the 6 loci tested and were consistent with their MSI-H status. In the so called MSS tumors and constitutive tissues (normal colon tissue, and PBLs) of all the HNPCC patients, low, but significant levels of MSI were seen in at least two of the loci studied. This phenomenon was not seen in the sporadic MSI constitutive tissues nor the normal controls and suggests haploinsufficiency, gain-of-function, or a dominant/negative basis of the instability in HNPCC patients carrying germline mutations for tumor suppressor genes. A different frequency and spectrum of mutant fragments suggests a different genetic basis (other than a major mutation in MLH1 or MSH2) for disease in MSI-L and MSS HNPCC patients. ^

A role for p53 in sensing DNA damage and triggering apoptotic responses to anticancer agents

The p53 tumor suppressor protein plays a major role in cellular responses to anticancer agents that target DNA. DNA damage triggers the accumulation of p53, resulting in the transactivation of genes, which induce cell cycle arrest to allow for repair of the damaged DNA, or signal apoptosis. The exact role that p53 plays in sensing DNA damage and the functional consequences remain to be investigated. The main goal of this project was to determine if p53 is directly involved in sensing DNA damage induced by anticancer agents and in mediating down-stream cellular responses. This was tested in two experimental models of DNA damage: (1) DNA strand termination caused by anticancer nucleoside analogs and (2) oxidative DNA damage induced by reactive oxygen species (ROS). Mobility shift assays demonstrated that p53 and DNA-PK/Ku form a complex that binds DNA containing the anticancer nucleoside analog gemcitabine monophosphate in vitro. Binding of the p53-DNA-PK/Ku complex to the analog-containing DNA inhibited DNA strand elongation. Furthermore, treatment of cells with gemcitabine resulted in the induction of apoptosis, which was associated with the accumulation of p53 protein, its phosphorylation, and nuclear localization, suggesting the activation of p53 to trigger apoptosis following gemcitabine induced DNA strand termination. The role of p53 as a DNA damage sensor was further demonstrated in response to oxidative DNA damage. Protein pull-down assays demonstrated that p53 complexes with OGG1 and APE, and binds DNA containing the oxidized DNA base 8-oxoG. Importantly, p53 enhances the activities of APE and OGG1 in excising the 8-oxoG residue as shown by functional assays in vitro. This correlated with the more rapid removal of 8-oxoG from DNA in intact cells with wild-type p53 exposed to exogenous ROS stress. Interestingly, persistent exposure to ROS resulted in the accelerated onset of apoptosis in cells with wild-type p53 when compared to isogenic cells lacking p53. Apoptosis in p53+/+ cells was associated with accumulation and phosphorylation of p53 and its nuclear localization. Taken together, these results indicate that p53 plays a key role in sensing DNA damage induced by anticancer nucleoside analogs and ROS, and in triggering down-stream apoptotic responses. This study provides new mechanistic insights into the functions of p53 in cellular responses to anticancer agents. ^

Gain-of-function of the p53R172H mutation in a mouse model of Li -Fraumeni syndrome

Missense mutations in the p53 tumor-suppressor gene are the most common alterations of p53 in somatic tumors and in patients with Li-Fraumeni syndrome. p53 missense mutations occur in the DNA binding region and disrupt the ability of p53 to activate transcription. In vitro studies have shown that some p53 missense mutants have a gain-of-function or dominant-negative activity. ^ The p53 175 Arg-to-His (p53 R175H) mutation in humans has been shown to have dominant-negative and gain-of-function properties in vitro. This mutation is observed in the germline of individuals with Li-Fraumeni syndrome. To accurately model Li-Fraumeni syndrome and to examine the mechanistic nature of a gain-of-function missense mutation on in vivo tumorigenesis, we generated and characterized a mouse with the corresponding mutation, p53 R172H. p53R172H homozygous and heterozygous mice developed similar tumor spectra and survival curves as p53 −/− and p53+/− mice, respectively. However, tumors in p53+/R172H mice metastasized to various organs with high frequency, suggesting a gain-of-function phenotype by p53R172H in vivo. Mouse embryonic fibroblasts (MEFs) from p53R172H mice also showed gain-of-function phenotypes in cell proliferation, DNA synthesis, and transformation potential, while cells from p53+/− and p53−/− mice did not. ^ To mechanistically characterize the gain-of-function phenotype of the p53R172H mutant, the role of p53 family members, p63 and p73, was analyzed. Disruption of p63 and p73 by siRNAs in p53 −/− MEFs increased transformation potential and reinitiated DNA synthesis to levels observed in p53R172H/R172H cells. Additionally, p63 and p73 were bound and functionally inactivated by p53R172H in metastatic p53 R172H tumor-derived cell lines, indicating a role for the p53 family members in the gain-of-function phenotype. This study provides in vivo evidence for the gain-of-function effect of p53 missense mutations and more accurately models the Li-Fraumeni syndrome. ^

Function of GCIP (CCNDBP1), a helix -loop -helix leucine -zip protein, in cell differentiation and tumorigenesis

Cell growth and differentiation are complex and well-organized processes in which cells respond to stimuli from the environment by carrying out genetic programs. Transcription factors with helix-loop-helix (HLH) motif play critical roles in controlling the expression of genes involved in lineage commitment, cell fate determination, proliferation and tumorigenesis. This study has examined the roles of GCIP (CCNDBP1) in cell differentiation and tumorigenesis. GCIP is a recently identified HLH-leucine zipper protein without a basic region like the Id family of proteins. However, GCIP shares little sequence homology with the Id proteins and has domains with high acidic amino acids and leucine-rich regions following the HLH domain like c-Myc. Here we firstly demonstrate that GCIP is a transcription regulator related to muscle differentiation program. Overexpression of GCIP in C2C12 cells not only promotes myotube formation but also upregulates myogenic differentiation biomarkers, including MHC and myogenein. On the other hand, our finding also suggests that GCIP is a potential tumor suppressor related to cell cycle control. Expression of GCIP was significantly down-regulated in colon tumors as compared to normal colon tissues. Overexpression of GCIP in SW480 colon cancer cell line resulted in a significant inhibition on tumor cell colony formation on soft agar assays while silencing of GCIP expression by siRNA can promote cell proliferation and colony formation. In addition, results from transgenic mice specifically expressing GCIP in liver also support the idea that GCIP is involved in the early stage of hepatocarcinogenesis and decreased susceptibility to chemical hepatocarcinogenesis. ^