The F-box protein FBXO44 mediates BRCA1 ubiquitination and degradation.

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF(FBXO44)) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF(FBXO44) reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF(FBXO44) is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF(FBXO44)-mediated BRCA1 degradation might contribute to sporadic breast tumor development.

Downregulation and growth inhibitory role of FHL1 in lung cancer.

Four and a half Lin-11, Isl-1, Mac-3 (LIM) protein 1 (FHL1) has been linked to carcinogenesis. However, the role of FHL1 in lung cancer remains unclear and the detailed mechanism underlying its tumor suppressive role is poorly understood. The purpose of this study was to examine FHL1 expression in lung cancer patients and to investigate how it was associated with lung cancer cell growth. Immunoblotting and immunohistochemistry showed that FHL1 protein was downregulated in over 90% of 80 lung cancer patients. FHL1 expression was strongly correlated with tumor histological types (p < 10(-4) ) and the differentiation of the tumor (p = 0.002). FHL1 inhibited anchorage-dependent and -independent growth of human lung cancer cell lines. The inhibitory effects of FHL1 on lung cancer cell growth were associated with both the G1 and the G2/M cell cycle arrest concomitant with a marked inhibition of cyclin A, cyclin B1 and cyclin D as well as the induction of the cyclin dependent kinase inhibitors p21 (WAF1/CIP1) and p27 (Kip1). Direct intratumoral injection of an adenovirus expressing FHL1 dramatically suppressed the growth of A549 lung cancer cells in nude mice. Our data suggest that reduced expression of FHL1 may play an important role in the development and progression of lung cancer and that FHL1 may be a useful target for lung cancer gene therapy.

Human four-and-a-half LIM family members suppress tumor cell growth through a TGF-beta-like signaling pathway.

The four-and-a-half LIM (FHL) proteins belong to a family of LIM-only proteins that regulate cell proliferation, differentiation, and apoptosis. The exact functions of each FHL protein in cancer development and progression remain unknown. Here we report that FHL1, FHL2, and FHL3 physically and functionally interact with Smad2, Smad3, and Smad4, important regulators of cancer development and progression, in a TGF-beta-independent manner. Casein kinase 1delta, but not the TGF-beta receptor, was required for the FHL-mediated TGF-beta-like responses, including increased phosphorylation of Smad2/3, interaction of Smad2/3 and Smad4, nuclear accumulation of Smad proteins, activation of the tumor suppressor gene p21, and repression of the oncogene c-myc. FHL1-3 inhibited anchorage-dependent and -independent growth of a human hepatoma cell line in vitro and tumor formation in nude mice. Further analysis of clinical samples revealed that FHL proteins are often downregulated in hepatocellular carcinomas and that this correlates with decreased TGF-beta-like responses. By establishing a link between FHL proteins and Smad proteins, this study identifies what we believe to be a novel TGF-beta-like signaling pathway and indicates that FHL proteins may be useful molecular targets for cancer therapy.

Hepatitis B virus X protein and the estrogen receptor variant lacking exon 5 inhibit estrogen receptor signaling in hepatoma cells.

Hepatitis B virus (HBV) X protein (HBx) is considered to play a role in the development of hepatocellular carcinoma (HCC) during HBV infection. HCC was shown to be more prevalent in men than in women. Estrogen, which exerts its biological function through estrogen receptor (ER), can inhibit HBV replication. ERDelta5, an ERalpha variant lacking exon 5, was found to be preferentially expressed in patients with HCC compared with patients with normal livers. Here, we report the biological role of ERDelta5 and a novel link between HBx and ERalpha signaling in hepatoma cells. ERDelta5 interacts with ERalpha in vitro and in vivo and functions as a dominant negative receptor. Both ERalpha and ERDelta5 associate with HBx. HBx decreases ERalpha-dependent transcriptional activity, and HBx and ERDelta5 have additive effect on suppression of ERalpha transactivation. The HBx deletion mutant that lacks the ERalpha-binding site abolishes the HBx repression of ERalpha. HBx, ERalpha and histone deacetylase 1 (HDAC1) form a ternary complex. Trichostatin A, a specific inhibitor of HDAC enzyme, can restore the transcriptional activity of ERalpha inhibited by HBx. Our data suggest that HBx and ERDelta5 may play a negative role in ERalpha signaling and that ERalpha agonists may be developed for HCC therapy.

Potentiation of Smad-mediated transcriptional activation by the RNA-binding protein RBPMS.

Smad2, Smad3 and Smad4 proteins are considered to be key mediators of transforming growth factor-beta (TGF-beta) signaling. However, the identities of the Smad partners mediating TGF-beta signaling are not fully understood. Here, we show that RNA-binding protein with multiple splicing (RBPMS), a member of the RNA-binding protein family, physically interacts with Smad2, Smad3 and Smad4 both in vitro and in vivo. The presence of TGF-beta increases the binding of RBPMS with these Smad proteins. Consistent with the binding results, overexpression of RBPMS enhances Smad-dependent transcriptional activity in a TGF-beta-dependent manner, whereas knockdown of RBPMS decreases this activity. RBPMS interacts with TGF-beta receptor type I (TbetaR-I), increases phosphorylation of C-terminal SSXS regions in Smad2 and Smad3, and promotes the nuclear accumulation of the Smad proteins. Moreover, RBPMS fails to enhance the transcriptional activity of Smad2 and Smad3 that lack the C-terminal phosphorylation sites. Our data provide the first evidence for an RNA-binding protein playing a role in regulation of Smad-mediated transcriptional activity and suggest that RBPMS stimulates Smad-mediated transactivation possibly through enhanced phosphorylation of Smad2 and Smad3 at the C-terminus and promotion of the nuclear accumulation of the Smad proteins.

[Effects of trichostatin A on the interaction between HBx and histone deacetylase protein 1].

OBJECTIVES: To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1). METHODS: Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo. RESULTS: Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1. CONCLUSIONS: HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

The antibody preparation and expression of human Pescadillo.

To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigenesis, the cDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated. Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells, and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer, colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a potential target in cancer diagnosis and therapy.

[An analysis of the features of HBx protein distributed in liver cells and its expression in E. coli].

OBJECTIVE: To investigate the features of HBx protein distributed in liver cells and its expression in E. coli. METHODS: The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography. RESULTS: The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded. CONCLUSION: This study may provide a basis for further study on the biological function of HBx at the protein level.

[Malignant transformation of NIH3T3 cells induced by ectopic expression of PC-1 gene].

OBJECTIVE: To establish a mouse fibroblastic cell line stably transfected with PC-1 gene, and using such cell line to investigate tumor development and progression imposed by the ectopic expression of PC-1 gene. METHODS: Eukaryotic expression vector pcDNA3.1(-)/myc-his-pc-1 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfectants were selected by G418. The integration and expression of ectopic PC-1 gene were analyzed by PCR and RT-PCR. Cytomorphological analysis, MTT, soft agar colony formation and nude mice tumorigenesis assay were used to evaluate the effects of PC-1 gene expression on tumor development and progression. RESULTS: NIH 3T3 cell lines stably expressing PC-1 gene were successfully established and confirmed by PCR and RT-PCR analyses of the integration and expression of ectopic PC-1 gene. Comparing with the parental cell line and cells transfected with control vector, the PC-1 gene transfectants acquired several phenotypes of transformed cells: increasing growth rate, ability to grow and form cell colonies on soft agar, and becoming tumorigenic in nude mice. CONCLUSION: Ectopic expression of PC-1 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in vivo.

[A novel system for characterization of the transcription activating proteins in mammalian cells].

A system used for detecting the transcriptional activating activity of the function-unknown gene products in mammalian cells was developed. Based on the plasmid pTet-Off and the eukaryotic expressing vector pCDNA3.1B(-)/myc-his, firstly, we constructed a set of recombinant plasmids namely pZHO1 (for cloning into the foreign gene fragment and as a negative control), pZHO2 (as a positive control). The system also includes the plasmids pTRE-luc (encoding the Firefly luciferase reporter gene) and pRL-TK (encoding Renilla luciferase gene as background control). To confirm the feasibility of the system, the plasmids pZHO1, pZHO2 and pZHO3 (encoding p53 transcriptional activating domain, containing 73 amino acids in its N terminal) was contransfected into such mammalian cells as C4-2, MCF-7, COS7 respectively, each with pTRE-luc and pRL-TK plasmids, the feasibility of the system was determined by comparing the relative activity of Firefly luciferase activity ratio of and Renilla in different transfecting panels. Our research result showed that the system we constructed can be used for detecting the transcriptional activating activity of the target protein molecules in mammalian cells.

PES1 promotes breast cancer by differentially regulating ERα and ERß.

The initiation of breast cancer is associated with increased expression of tumor-promoting estrogen receptor α (ERα) protein and decreased expression of tumor-suppressive ERß protein. However, the mechanism underlying this process is unknown. Here we show that PES1 (also known as Pescadillo), an estrogen-inducible protein that is overexpressed in breast cancer, can regulate the balance between ERα and ERß. We found that PES1 modulated many estrogen-responsive genes by enhancing the transcriptional activity of ERα while inhibiting transcriptional activity of ERß. Consistent with this regulation of ERα and ERß transcriptional activity, PES1 increased the stability of the ERα protein and decreased that of ERß through the ubiquitin-proteasome pathway, mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). Moreover, PES1 transformed normal human mammary epithelial cells and was required for estrogen-induced breast tumor growth in nude mice. Further analysis of clinical samples showed that expression of PES1 correlated positively with ERα expression and negatively with ERß expression and predicted good clinical outcome in breast cancer. Our data demonstrate that PES1 contributes to breast tumor growth through regulating the balance between ERα and ERß and may be a better target for the development of drugs that selectively regulate ERα and ERß activities.

Tissue type-specific modulation of ER transcriptional activity by NFAT3.

NFAT3 belongs to the NFAT family of transcription factors playing important roles in the development of several organ systems and was found to act as a transcriptional coactivator of estrogen receptors (ERalpha and ERbeta) in breast cancer cells. Since some cofactors of transcription factors show cell or tissue type-specific effects on transcriptional regulation, we investigated the effect of NFAT3 on the transcriptional activity of ERs in different cell lines originated from kidney. Surprisingly, overexpression of NFAT3 in these cell types decreased dose-dependently both ERalpha and ERbeta transcriptional activities in a ligand-independent manner. Knockdown of endogenous NFAT3 using NFAT3 small interfering RNA (siRNA) increased ER transcriptional activities. NFAT3 deletion mutants lacking the ER-binding sites completely abolished the NFAT3 repression of ERalpha and ERbeta transcriptional activities. Replacement of Ser168 and Ser170, the amino acid residues on which NFAT3 can be phosphorylated, with Ala did not change the ability of NFAT3 to inhibit the transcriptional activity of ERalpha and ERbeta. Taken together, these results demonstrate that NFAT3 is a new kind of cofactor that displays dual transcription modulation mode dependent on tissue types.

[Preparation of antibody against Memo protein and analysis of expression profile of Memo in mouse tissues].

AIM: To prepare and characterize antibody against Memo protein and to detect the tissue distribution of Memo in mice. METHODS: Fusion protein GST-Memo was expressed and purified, and polyclonal antibody against Memo was prepared by immunizing mice. A FLAG-tagged eukaryotic expression vector pcDNA3-FLAG-Memo was constructed. The specificity of the antibody was detected by Western blot. RESULTS: An eukaryotic expression vector pcDNA3-FLAG-Memo was obtained. The polyclonal antibody was found to be specific to Memo. Memo protein was widely expressed in mouse tissues using the obtained antibody in Western blot. CONCLUSION: Antibody specific to Memo has been successfully obtained, which provides useful tool for investigation into Memo-associated mechanisms of tumor metastasis and invasiveness.

[Construction of recombinant plasmid using Neo/E Technology].

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.

[Cloning and Expression of Various Deletants of Gsalpha Gene in Escherichia Coli]

Gsalpha gene mutation has been discovered in some human tumors. In our previous studies, three novel deletants of Gsalpha gene, Gsalpha L-1(500 bp), Gsalpha L-2(300 bp), and Gsalpha L-3(200 bp), and wild type Gsalpha-4(1 200 bp) were found in human leukemia cell lines and detected in leukemic cells from patients with acute leukemia. To investigate the construction, function and biological significance of the deletants, the plasmids of Gsalpha L-1, Gsalpha L-2 and wild Gsalpha-4 were transformed into E. coli DH5, amplified by PCR, and cloned in expression vector pET22b(+), and then transformed into E. coli, respectively. As a result, higher levels of expression of three recombinants were obtained in form of inclusion bodies. The results suggested that these Gsalpha isoforms have an open reading frame of gene and can be expressed in vitro. The data lay a foundation to study the relation of Gsalpha gene to leukemogenesis.

[Expression of PC-1 gene in tumor tissues, cloning and analysis of its putative promoter region].

BACKGROUND & OBJECTIVE: PC-1 is a novel gene which is overexpressed in bone metastasis and androgen independent prostate cancer cell line C4-2. The objective of this study was to evaluate the expression level of PC-1 gene in multiple human tumor and normal tissues, and clone a series of putative PC-1 gene promoter regions and analyze their promoter activities. METHODS: PC-1 gene specific DNA sequence was used as probe to hybridize with total RNA from 10 pairs of tumor and normal tissues; The C4-2 cell genomic DNA was used as a template in the polymerase chain reaction to amplify PC-1 upstream regions from the translation initiation codon. The PCR product was directly cloned into the luciferase reporter vector pGL3-basic. C4-2 cells were transiently transfected with above recombinant plasmids and the putative promoter activities were analyzed by luciferase assay. RESULTS: PC-1 gene expression level is remarkable higher in multiple tumor tissues than in their matched normal tissues; there is no promoter activity in the 340 bp fragment from the upstream of initiation codon, while 1099 bp, 1337 bp, 1579 bp, 1831 bp, and 4939 bp fragments had the promoter activities. CONCLUSION: The PC-1 gene expression is specifically activated in multiple human tumor tissues, suggesting that PC-1 gene expression might be involved in cancer development. Our primary data shows that the highest promoter activity of PC-1 gene is within the 4939 bp DNA fragment and maybe there exists an enhancer element between the 1831 bp and 4939 bp area.

[Induction of protective immunity in rhesus monkey by inoculation with recombinant fusion protein of cholera toxin B subunit-multivalent epitopes of Plasmodium falciparum].

Rhesus monkeys (5 in each group) were inoculated with recombinant fusion protein of cholera toxin B subunit and multi-valent epitopes of Plasmodium falciparum intranasal or intramuscular (i.m.). Immune-responses and protective effect were evaluated. The antibody titer (Geometry mean) against CTB reached 1:512 (intranasal) and 1:10000 (i.m.) 14 day after 3rd immunization, and antibodies against P. falciparum were also elucidated, the titers in i.m. group were also significantly higher than that in intranasal group. The monkeys were challenged with 1.25 x 10(8) sporozoites of P. cynomolgi, Patent infection was observed in all 5 monkeys in control group inoculated with PBS in 10 - 14 days after challenge. Patent infection was also observed in 5 animals inoculated via intranasal and 2 animals in intramuscular group 19th days after challenge, But the infection last only 4 days in 3 animals in intranasal group and 2 animals in intramuscular group. The results demonstrated that the vaccine candidate could induce protective immune-responses in rhesus monkey against the challenge of P. cynomolgi.