927 resultados para Geography|Health Sciences, Public Health|Health Sciences, Epidemiology|Health Sciences, Oncology
Resumo:
The major risk factors for liver cancer in Southeast Asia: HBV infection, aflatoxin exposure and p53 expression/mutation, were examined in experimental models. Four groups were examined for development of hepatocellular carcinoma (HCC) with and without neonatal exposure to aflatoxin (AFB$\sb1)$: (Group I.) Transgenic HBsAg mice with one p53 allele. (Group II) Transgenic HBsAg mice with two p53 alleles. (Group III) Non-transgenic litter mates with one p53 allele. (Group IV) Non-transgenic litter mates with two p53 alleles. HCC developed in Group I animals exposed to aflatoxin at an earlier time and were of a higher grade than those seen later in other groups. These results provide an explanation for as to why p53 is a target for deletion and/or mutation in human HCC especially when found in high risk areas where HBV infection and Aflatoxin B1 food contamination is high, and nicely illustrates a synergistic interaction among these three factors. None of the tumors analyzed had loss or mutation in the p53 gene.^ To determine the significance of the specific p53ser249 mutation found in HBV/aflatoxin associated human hepatomas in an in-vivo experimental model using transgenic mice, a two-nucleotide change in the mouse p53 gene at amino acid position 246, which is equivalent to that of 249 in human p53, was introduced. Transgenic mice with mutant p53 controlled by the albumin promoter were generated and shown to express the p53ser246 mutant RNA and protein specifically in liver. Three groups were examined for development of HCC with and without neonatal exposure to aflatoxin: (Group V) Transgenic p53ser246 mice with two p53 alleles. (Group VI) Transgenic p53ser246 mice with one p53 allele. (Group VII) Double transgenic for p53ser246 and HBsAg with two p53 alleles. One hundred percent of male mice with the three risk factors injected with aflatoxin developed high grade liver tumors, compared to 66.6% from group VI and only 14.2% of group V suggesting synergistic interaction between HBsAg and this particular ser246 p53 mutation.^ In order to examine the growth properties of hepatocytes and correlation with p53 loss and/or mutation, cell proliferation and ploidy analysis of liver from normal heterozyous, homozygous null mice and from transgenic mutant p53ser246, mice were studied. Loss of wild-type p53 increased G1/G0 ratios of cells as well as proliferation and decreased cell ploidy. The mutant p53ser246 did not show a significant effect on cell ploidy or proliferation. However a striking 5-10X increase in G1/G0 ratio suggests that this specific mutation specifically induces G0 to G1 transition, which in turn further predisposes hepatocytes to the damaging effect of Aflatoxin. (Abstract shortened by UMI.) ^
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A combination of psoralen and ultraviolet-A radiation, commonly referred to as "PUVA," is widely used in the treatment of psoriasis. However, PUVA treatment increases the risk of developing skin cancer in psoriasis patients and induces skin cancer in mice. It is, however unknown whether the increased incidence of skin cancer in PUVA treated psoriasis patients is due to the carcinogenic effects of PUVA therapy or due to an indirect effect such as immunosuppression, which can permit the growth of tumors induced by UVB radiation. In this study, we used the p53 tumor suppressor gene as a molecular marker to determine whether PUVA-induced mouse skin cancers contain unique mutations in p53 that are different from UV-induced mutations, and if so, determine whether skin cancers from PUVA treated patients have PUVA-type or UV-type p53 mutations. Since the DNA lesions induced by PUVA are quite different from those induced by UV, we hypothesize that p53 mutations induced by PUVA may also be different from those induced by UV.^ Analysis of PUVA-induced murine skin cancers for p53 mutations revealed that 14 of 15 (93%) missense mutations detected in these cancers were localized at 5$\sp\prime$-TA/5$\sp\prime$-TAT sites, potential sites of psoralen photoadditions. Mutations at these sequences are exceedingly rare in UV-induced murine skin cancers. In addition, PUVA-induced murine skin cancers did not contain UV signature (C $\to$ T or CC $\to$ TT transitions) mutations in p53. These results suggest that PUVA induces unique mutations in p53 that can be distinguished from those induced by UV.^ Next we determined whether SCCs arising in PUVA treated psoriasis patients have PUVA-type or UV-type p53 mutations. The results indicated that 16 of 25 (64%) missense p53 mutations detected in SCCs from PUVA treated patients were located at 5$\sp\prime$-TG, 5$\sp\prime$-TA and 5$\sp\prime$-TT sites, putative sites of psoralen photobinding. Interestingly, about 32% of p53 mutations detected in SCCs from PUVA treated patients had the UV signature. Taken together these results suggest that both PUVA and UVB play a role in the development of SCCs in psoriasis patients undergoing PUVA therapy. ^
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Follicular lymphoma is the most common lymphoid malignancy in humans. The bcl-2 transgenic mice, which mimic the human follicular lymphoma, initially exhibit a polyclonal hyperplasia due to the overriding of apoptosis by deregulated bcl-2. After a latency period of 15 month 20% of the animals developed clonal lymphomas. Approximately, 50% of these high grade lymphomas presented chromosomal translocations involving c-myc, suggesting that deregulation of this gene is important in the complementation with bcl-2. E$\mu$-myc x bcl-2 double transgenic mice were generated to assess the ability of this two genes to complement in an in vivo system. The double transgenic mice presented a shortened latency (3-4 weeks) and higher incidence of tumor development. Quantification of the extent of programmed cell death indicated that bcl-2 can abrogate the high rate of apoptotic cell death that results from myc deregulation. Bcl-2-Ig, E$\mu$-myc, and bcl-2/E$\mu$-myc lymphomas were examined using PCR-SSCP to detect the presence of p53 mutations in exons 5-9. A high incidence of p53 mutations in E$\mu$-myc lymphomas suggested that inactivating lesions of p53 may represent an important step in the genetic complementation of c-myc in lymphomagenesis. Surprisingly, p53 mutations were quite uncommon in bcl-2 lymphomas suggesting that inactivating mutations of p53 and overexpression of bcl-2 may not cooperate in lymphoma progression. To assess this question, we generated mice that contained a deregulated bcl-2 gene and were nullizygous for p53 (p53KO). No reduction in the tumor latency was observed in the p53KO/bcl-2-Ig hybrid mice when compared with p53 KO mice. Using splenic mononuclear cells isolated from p53KO mice and bcl-2 transgenic mice we demonstrated that bcl-2 suppresses p53 mediated apoptosis in response to DNA damage initiated by $\gamma$-radiation even though p53 protein is induced normally in the bcl-2 overexpressing cells. Western analysis of the expression of p53 target proteins after $\gamma$-radiation showed a correlation between the p53-dependent induction of bax protein after radiation and the ability of p53 to mediate apoptosis. ^
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The purpose of this study was to investigate the role of the c-KIT receptor in the progression of human melanoma and the mechanism(s) for the regulation of c-KIT gene expression in human melanoma.^ The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) (metastatic phenotype) are not well-defined. Expression of the tyrosine-kinase receptor c-KIT progressively decreases during local tumor growth and invasion of human melanomas. To provide direct evidence that the metastasis of human melanoma is associated with the loss of c-KIT expression, highly metastatic A375SM cells, which express very low or undetectable levels of c-KIT, were tranduced with the human c-KIT gene. We demonstrated that enforced c-KIT expression in highly metastatic human melanoma cells significantly suppressed their tumorigenicity and metastatic propensity in nude mice. In addition, we showed that the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. These results suggest that loss of c-KIT receptor may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, thus contributing to tumor growth and eventually metastasis.^ Furthermore, we investigated the possible mechanism(s) for the down-regulation of c-KIT gene expression in malignant melanoma. Sequence analysis of the c-KIT promoter indicated that this promoter contains several consensus binding-site sequences including three putative AP2 and two Myb sites. Although Myb was shown to be associated with c-KIT expression in human hemotopoietic cells, we found no correlation between c-KIT expression and Myb expression in human melanoma cell lines. In contrast, we showed that c-KIT expression directly correlates with expression of AP2 in human melanoma cells. We found that highly metastatic cells do not express the transcription factor AP2. Expression of AP2 in A375SM cells (c-KIT-negative and AP2-negative) was enough to restore luciferase activity driven by the c-KIT promoter in a dose-dependent manner. On the other hand, co-expression of the dominant-negative form of AP2 (AP2B) in Mel-501 cells (c-KIT-positive and AP2-positive) resulted in two-fold reduction in luciferase activity. Electrophoretic mobility shift assays revealed that the c-KIT promoter contains functional AP2 binding sites which could associate with AP2 protein. Endogenous c-KIT gene expression levels were elevated in AP2 stably-transfected human melanoma A375SM cells. Expression of exogenous AP2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. The c-KIT ligand, SCF, also induced apoptosis in the AP2 stably-transfected A375SM cells. The identification of AP2 as an important regulator for c-KIT expression suggests that AP2 may have tumor growth and metastasis inhibitory properties, possibly mediated through c-KIT/SCF effects on apoptosis of human melanoma cells. Since AP2 binding sites were found in the promoters of other genes involved in the progression of human melanoma, such as MMP2 (72 kDa collagenase), MCAM/MUC18 and P21/WAF-1, our findings suggest that loss of AP2 expression might be a crucial event in the development of malignant melanoma. ^
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The adenovirus type 5 E1A gene products have numerous functions in cells, which serve as useful tools in studying the mechanisms of either oncogenesis or tumor suppression. To understand the mechanisms of E1A-mediated tumor suppression, we introduced an Ad5 E1A gene into murine melanoma cells, and characterized E1A-mediated biological functions both in vitro and in vivo. The results of the study indicated that: (i) Ad5 E1A mediated tumor suppression in rodent tumor cells; (ii) E1A-mediated tumor suppression is associated with E1A-mediated apoptosis in vivo.^ To determine which functional region(s) of E1A is(are) required for E1A-mediated apoptosis and whether E1A-mediated apoptosis is required for E1A-mediated tumor suppression, we established stable transfectants of E1A mutants, which have deletion mutation at either the N-terminal (p300-binding) or the CR2 (pRb-binding) domain or both, and then characterized biological functions both in vitro and in vivo. The results of the study indicate that the CR2 domain of E1A is required for E1A-mediated apoptosis, while the N-terminal domain of E1A is dispensable. Interestingly, either of the two domains is able to mediate tumor suppression, since mutant E1A with a single deletion at either domain still suppressed tumor growth. Importantly, deletion mutations at both the N-terminal and the CR2 domains of E1A abrogated E1A-mediated tumor suppression, suggesting both regions are required for E1A-mediated tumor suppression. The results demonstrate that E1A-mediated apoptosis is not the only mechanism for E1A-mediated tumor suppression. Thus, the N-terminal and CR2 domains of E1A mediated two independent mechanisms of tumor suppression.^ To understand the mechanism of E1A-mediated apoptosis, we examined the temporal relationship of molecular events during the apoptotic cascades after UV radiation and serum depletion in both the E1A-expressing cells and parental cells. Kinetic analysis of JNK activity indicates that the JNK pathway is greatly increased in response to UV light in E1A transfectants, suggesting that extracellular stress stimuli have been converted into intracellular stress signals with greater magnitude in E1A transfectants than those in parental cells. Thus, E1A-mediated sensitization precedes these events. As ceramide has been proposed as second messenger and upstream activator of JNK pathway for stress-induced apoptosis, we also examined the roles of ceramide in apoptosis and the relationship with JNK pathway. The results indicate that E1A transfectants do not have increased sensitivity to ceramide. Therefore, E1A-mediated sensitization to UV radiation cannot be attributed to an increased sensitivity to ceramide. Furthermore, UV-induced JNK activation correlates with UV-induced apoptosis, while lethal dose of ceramide does not activate JNK. Thus, activation of JNK pathway is independent of the ceramide pathway. In addition, E1A transfectants also have increased activation of NF-kB in response to UV. These results suggest that E1A-mediated sensitization is an early event which associates with conversion of extracellular stress stimuli into amplified intracellular signals. The mechanism of E1A-mediated sensitization and its relationship with other pathways are discussed. ^
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HER-2/neu is a receptor tyrosine kinase highly homologous with epidermal growth factor receptor. Overexpression and/or amplification of HER-2/neu has been implicated in the genesis of a number of human cancers, especially breast and ovarian cancers. Transcriptional upregulation has been shown to contribute significantly to the overexpression of this gene. Studies on the transcriptional regulation of HER-2/neu gene are important for understanding the mechanism of cell transformation and developing the therapeutic strategies to block HER-2/neu-mediated cancers. PEA3 is a DNA binding transcriptional factor and its consensus sequence exists on the HER-2/neu promoter. To examine the role of PEA3 in HER-2/neu expression and cell transformation, we transfected PEA3 into the human breast and ovarian cancer cells that overexpress HER-2/neu and showed that PEA3 dramatically represses HER-2/neu transcription. PEA3 suppresses the oncogenic neu-mediated transformation in mouse fibroblast NIH 3T3 cells. Expression of PEA3 selectively blocks the growth of human cancer cells that overexpress HER-2/neu and inhibits their colony formation. It does not occur in the cancer cells expressing basal level of HER-2/neu. Further studies in the orthotopic ovarian cancer model demonstrated that expression of PEA3 preferentially inhibits growth and tumor development of human cancer cells that overexpress HER-2/neu, the tumor-bearing mice survived significantly longer if treated by injection of the PEA3-liposome complex intraperitoneally. Immunoblotting and immunohistochemical analysis of the tumor tissues indicated that PEA3 mediates the tumor suppression activity through targeting HER-2/neu-p185. Thus, PEA3 is a negative regulator of HER-2/neu gene expression and functions as a tumor suppressor gene in the HER-2/neu-overexpressing human cancer cells.^ The molecular mechanisms of PEA3 mediated transcriptional repression were investigated. PEA3 binds specifically at the PEA3 site on HER-2/neu promoter and this promoter-binding is required for the PEA3 mediated transcriptional repression. Mutation of the PEA3 binding site on HER-2/neu promoter causes decreased transcriptional activity, indicating that the PEA3 binding site is an enhancer-like element in the HER-2/neu-overexpressing cells. We therefore hypothesized that in the HER-2/neu-overexpressing cells, PEA3 competes with a transactivator for binding to the PEA3 site, preventing the putative factor from activating the transcription of HER-2/neu. This hypothesis was supported by the data which demonstrate that PEA3 competes with another nuclear protein for binding to the HER-2/neu promoter in vitro, and expression of a truncated protein which encodes the DNA binding domain of PEA3 is sufficient to repress HER-2/neu transcription in the HER-2/neu-overexpressing human cancer cells. ^
Resumo:
c-Src, a protein tyrosine kinase (PTK) the specific activity of which is increased $>$20-fold in $\sim$80% of colon tumors and colon tumor cell lines, plays a role in both growth regulation and tumorigenicity of colon tumor cells. To examine the effect of increased c-Src specific activity on colon tumor cells, coumarin-derived tyrosine analog PTK inhibitors were assessed in a standard colon tumor cell line, HT-29. Of the nine compounds tested for inhibiting c-Src activity in a standard immune complex kinase assay from c-Src precipitated from HT-29 cells, the 7,8-dihydroxy-containing compounds daphnetin and fraxetin were most effective, with IC$\sb{50}$s of 0.6 $\pm$ 0.2 mM and 0.6 $\pm$ 0.3 mM, respectively. Treatment of HT-29 cells with daphnetin resulted in inhibition of cell growth in a dose-dependent manner. In contrast, scopoletin, a relatively poor Src inhibitor in vitro, did not inhibit HT-29 cell growth in the concentration range tested. In daphnetin treated cells, a dose-dependent decrease of c-Src activity paralleling cell growth inhibition was also observed; the IC$\sb{50}$ was 0.3 $\pm$ 0.1 mM for c-Src autophosphorylation. In contrast, the IC$\sb{50}$ for c-Src protein level was $>$ 0.6 mM, indicating that the effects of daphnetin were primarily an enzymatic activity of c-Src, rather than protein level in HT-29 cells. These results are the first to demonstrate that c-Src specific activity regulates colon tumor cell growth.^ To elucidate the signaling pathways activated by c-Src in colon tumor cells, the Src family substrate FAK, which has been shown to play a role in both extracellular matrix-dependent cell growth and survival, was examined. Coprecipitation assays showed Src-FAK association in detergent insoluble fractions of both attached and detached HT-29 cells, indicating that Src-FAK association in HT-29 cells is stable and, unlike untransformed cells, not dependent on cell-substratum contact. FAK also coprecipitated with Grb2, an adaptor protein also playing a role in cell proliferation and survival, in both attached and detached HT-29 cells, suggesting that a Src-FAK-Grb2-mediated signaling pathway(s) in HT-29 cells is/are constitutively activated.^ FAK was also analyzed in c-src antisense HT-29 clones AS15 and AS33 in which c-Src is specifically reduced by transfection of an antisense expression vector. FAK protein level is unexpectedly decreased in both AS15 and AS33 cells by 5-fold and 1.5-fold compared to HT-29, respectively, corresponding with the decreased expression of c-Src observed in these cells. FAK protein level was not decreased compared to parental in the c-src "sense" clone S8. Northern blot analyses showed decreased FAK mRNA levels compared to parental in AS15 and AS33, correlating with decreased FAK protein level, indicating that FAK activity in the antisense cells is regulated, at least in part, by altering FAK expression, and that this regulation is Src dependent. Because FAK has been implicated in anoikis, the ability of c-src antisense cells to survive in the absence of cell-substratum contact was examined. Decreased cell survival is seen in both AS15 and AS33, correlating with the decreases in c-Src and FAK levels and tumorigenicity in these cells. These results suggest that at least one mechanism by which activation of c-Src contributes to tumorigenic phenotype of colon tumor cells is by aberrantly promoting a survival signal through unregulated Src-FAK-Grb2 complexes. (Abstract shortened by UMI.) ^
Resumo:
Cellular oncogenes and tumor suppressor genes regulate cellular adhesion and proliferation, two important events in malignant transformation. Even though receptor-like protein tyrosine phosphatases (R-PTPs) can influence these events, their role in malignant transformation has not been studied. The major goal of this study was to determine whether downregulation of R-PTP$\mu$ expression in lung epithelial cells is associated with or causal to neoplastic transformation. Examination of R-PTP$\mu$ expression in normal and carcinoma cells demonstrated that lung epithelial cells expressed R-PTP$\mu$ whereas lung carcinoma cells did not, and that incubation with TGF-$\alpha$ and HGF induced a two fold increase in R-PTP$\mu$ mRNA expression. To associate the expression of R-PTP$\mu$ with neoplastic transformation, we transfected lung epithelial cells with the H-ras oncogene. Transformation resulted in the activation of the MAPK signal transduction pathway, the hyperphosphorylation of c-met, and the production of HGF. Upon analysis of R-PTP$\mu$ expression, we observed a significant decrease in R-PTP$\mu$ mRNA and protein levels suggesting that transformation can directly or indirectly downregulate the expression of R-PTP$\mu.$ TGF-$\beta$ reversed the H-ras transformed phenotype, an event directly correlated with upregulation of R-PTP$\mu.$ To provide a casual relationship between R-PTP$\mu$ and cessation of tumor cell growth, we transfected carcinoma cells with the wild type R-PTP$\mu$ cDNA. Transiently expressing cells were selected by FACS using the mAb 3D7 and plated into individual wells. Carcinoma cells positive for R-PTP$\mu$ expression did not grow into colonies whereas non-R-PTP$\mu$ expressing carcinoma cells did, suggesting that expression of R-PTP$\mu$ arrested cell growth. To better understand the growth arrest induced by R-PTP$\mu$, we transfected the H-ras transformed lung epithelial cell line (MvLu-1-ras) with R-PTP$\mu$ (MvLu-1-ras/R-PTP$\mu$). Examination of growth factor receptor phosphorylation revealed significant inhibition of c-met and EGF-R. Furthermore, these cells underwent apoptosis in the absence of serum. Taken together the data demonstrate that the downregulation of R-PTP$\mu$ expression is an important step in neoplastic transformation of lung epithelial cells and that its presence can induce apoptosis and inhibit the signaling of c-met and EGF-R, two major growth factor receptors in lung carcinoma. In conclusion, the expression of R-PTP$\mu$ is inversely correlated with neoplastic transformation, growth and survival of tumor cells. ^
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The most common molecular alterations observed in prostate cancer are increased bcl-2 protein expression and mutations in p53. Understanding the molecular alterations associated with prostate cancer are critical for successful treatment and designing new therapeutic interventions. Hormone-ablation therapy remains the most effective nonsurgical treatment; however, most patients will relapse with hormone-independent, refractory disease. This study addresses how hormone-ablation therapy may increase bcl-2, develops a transgenic model to elucidate the role of bcl-2 multistep prostate carcinogenesis, and assesses how bcl-2 may confer resistance to cell death induction using adenoviral wild-type p53 gene therapy. ^ Two potential androgen response elements were identified in the bcl-2 promoter. Bcl-2 promoter luciferase constructs were transfected into the hormone- sensitive LNCaP prostate cell line. In the presence of dihydrotestosterone, the activity of one bcl-2 promoter luciferase construct was repressed 40% compared to control cells grown in charcoal-stripped serum. Additionally, it was demonstrated that both bcl-2 mRNA and protein were downregulated in the LNCaP cells grown in the presence DHT. This suggests that DHT represses bcl-2 expression through possible direct and indirect mechanisms and that hormone-ablation therapy may actually increases bcl-2 protein. ^ To determine the role of bcl-2 in prostate cancer progression in vivo, probasin-bcl-2 mice were generated where human bcl-2 was targeted to the prostate. Increased bcl-2 expression rendered the ventral prostate more resistant to apoptosis induction following castration. When the probasin-bcl-2 mice were crossed with TRAMP mice, the latency to tumor formation was decreased. The expression of bcl-2 in the double transgenic mice did not affect the incidence of metastases. The double transgenic model will facilitate the study of in vivo effects of specific genetic lesions during the pathogenesis of prostate cancer. ^ The effects of increased bcl-2 protein on wild-type adenoviral p53-mediated cell death were determined in prostatic cell lines. Increased bcl-2 protected PC3 and DU145 cell lines, which possess mutant p53, from p53-mediated cell death and reductions in cell viability. Bcl-2 did not provide the same protective effect in LNCaP cell line, which expresses wild-type p53. This suggests that the ability of bcl-2 to protect against p53-mediated cell death is dependent upon the endogenous status of p53. ^
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Shc proteins are implicated in coupling receptor tyrosine kinases to the mitogen-activated protein kinase (MAPK) pathway by recruiting Grb2/SOS to the plasma membrane. To better understand the role of Shc in oncogenesis brought about by point mutation activated neu (p185*), we transfected a Shc mutant (ShcΔCH1), which lacks the Grb2 binding site Y317 by deletion of collagen-homology domain 1, into p185*-transformed NIH3T3 cells. The cellular transformation phenotypes were found to be largely suppressed by expression of ShcΔCH1. This study indicates that Shc plays a critical role in mediating the oncogenical signals of p185*. Although ShcΔCH1 still retained another Grb2 binding site (Y239/240), we did not detect its physical association with Grb2. We also found that ShcΔCH1 could associate with p185*; however, this association did not interfere with the endogenous Shc-p185* interaction or the Shc-Grb2 interaction. In addition, p185*-mediated MAPK/Elk activation, PI3-K activation and Src activation likewise was not inhibited by ShcΔCH1 expression. Taken together, our current study clearly indicates that ShcΔCH1 suppresses the p185*-induced transformation, and that this suppression is mediated through a MAPK-independent and possibly PI3-K, Src-independent pathway. These results suggest that Shc may be involved in other unidentified signal pathways which are critical for p185*-induced cellular transformation besides the three pathways that we have studied. ^
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Mutations in the p53 tumor suppressor gene are found in over 50% of human tumors and in the germline of Li-Fraumeni syndrome families. About 80% of these mutations are missense in nature. In order to study how p53 missense mutations affect tumorigenesis in vivo, we focused on the murine p53 arg-to-his mutation at amino acid 172, which corresponds to the human hot spot mutation at amino acid 175. The double replacement procedure was employed to introduce the p53 R172H mutation into the p53 locus of ES cells and mice were generated. An additional 1bp deletion in the intron 2 splice acceptor site was detected in the same allele in mice. We named this allele p53R172HΔg. This allele makes a small amount of full length p53 mutant protein. ^ Spontaneous tumor formation and survival were studied in these mice. Mice heterozygous for the p53R172HΔg allele showed 50% survival at 17 months of age, similar to the p53+/− mice. Moreover, the p53R172HΔg/+ mice showed a distinct tumor spectrum: 55% sarcomas, including osteosarcoms, fibrosarcomas and angiosarcomas; 27% carcinomas, including lung adenocarcinomas, squamous cell carcinomas, hepatocellular carcinomas and islet cell carcinomas; and 18% lymphomas. Compared to the p53+/− mice, there was a clear increase in the frequency of carcinoma development and a decrease in lymphoma incidence. Among the sarcomas that developed, fibrosarcomas in the skin were also more frequently observed. More importantly, osteosarcomas and carinomas that developed in the p53R172HΔg/+ mice metastasized at very high frequency (64% and 67%, respectively) compared with less than 10% in the p53+/− mice. The metastatic lesions were usually found in lung and liver, and less frequently in other tissues. The altered tumor spectrum in the mice and increased metastatic potential of the tumors suggested that the p53R172H mutation represents a gain-of-function. ^ Mouse embryonic fibroblasts (MEFs) from the mice homozygous and heterozygous for the p53R172HΔg allele were studied for growth characteristics, immortalization potential and genomic instability. All of the p53R172HΔg /+ MEF lines are immortalized under a 3T3 protocol while under the same protocol p53+/− MEFs are not immortalized. Karyotype analysis showed a persistent appearance of chromosome end-to-end fusion in the MEFs both homozygous and heterozygous for the p53R172HΔg allele. These observations suggest that increased genomic instability in the cells may cause the altered tumor phenotypes. ^
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The p53 gene is known to be one of the most commonly mutated genes in human cancers. Many squamous cell carcinomas of the head and neck (SCCHNs) have been shown to contain nonfunctional p53 as well. The use of p53-mediated gene therapy to treat such cancers has become an intensive area of research. Although there have been varied treatment responses to p53 gene therapy, the role that endogenous p53 status plays in this response has not been thoroughly examined. Because of this, the hypothesis of this study examined the role that the endogenous p53 status of cells plays in their response to p53 gene therapy. To test this, an adenoviral vector containing p53 (p53FAd) was administered to three squamous cell carcinoma lines with varied endogenous p53. The SCC9 cell line demonstrates no p53 protein expression, the SCC4 cell line displays overexpression of a mutant p53 protein, and the 1986LN cell line displays low to no expression of wild-type p53 protein as a consequence of human papillomavirus infection. After treatment with p53FAd, the cells were examined for evidence of exogenous p53 expression, growth suppression, alterations in cellular proteins, G1 growth arrest, apoptosis, and differentiation state. Each cell line exhibited exogenous p53 protein. Growth suppression was seen most prominently in the SCC9 cells, to some extent in the 1986LN cells, and little was seen with the SCC4 cells. WAF1/p21 protein was induced in all three cell lines, while PCNA, bcl-2, and bax expression was not significantly affected in any of the lines. Apoptosis developed first in SCC9 cells, next in 1986LN cells, with little seen in the SCC4 cells. The SCC9 line was the only line to show significant GI growth arrest. No significant differences were observed in the overall expression of differentiation markers, aside from increased keratin 13 mRNA levels in all three lines indicating a possible tendency toward differentiation. This study indicates that the endogenous p53 status of squamous cell carcinomas appears to play a critical role in determining the response to p53 adenoviral gene therapy. ^
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SHP1 is a cytosolic protein tyrosine phosphatase that contains two SH2 domains. It is highly expressed in hematopoietic cells and expressed in normal epithelium at lower levels. While SHP1 in hematopoietic cells is thought to be a negative regulator of cellular signaling by associating with and dephosphorylating various receptors and their downstream effectors after they become activated, its precise function in epithelium remains to be understood. The potential involvement of SHP1 in human tumorigenesis has been hypothesized from the findings that SHP1 can interact with, dephosphorylate, and regulate the activity of several protein tyrosine kinases (PTKs) implicated in human cancer. These PTKs include epidermal growth factor receptor (EGFR) and Src. Such speculation is also supported by the report that SHP1 is overexpressed in human ovarian cancers. ^ Here we report, for the first time, that the levels of SHP1 expression and activity are altered in human breast cancer cells in comparison with normal breast epithelium. In particular, SHP1 expression is nearly lost in the breast cancer cell lines MDA-MB231 and MDA-MB435. After the re-introduction of SHP1 both in wild type (wt) and enzymatically inactive (dn) forms, into the MDA-MB231 cells, we observed no changes in cellular proliferation. However, the overexpression of wt SHP1 led to increased anchorage-independent growth in the MDA-MB231 cells. SHP1 phosphatase activity is essential for such an increase since the overexpression of dn SHP1 had no effect. Enhanced turnorigenicity in nude mice was also observed in the MDA-MB231 cells overexpressing wt SHP1, but not dn SHP1, suggesting the crucial function of SHP1 enzymatic activity in this process. Our observations in this study indicate that SHP1 promotes tumorigenesis by a mechanism or mechanisms apart from enchancing angiogenesis. In addition, we have found no evidence that the overexpression of SHP1 could affect metastatic potential in the MDA-MB231 cells. ^ In the MDA-MB231 cells stably transfected with either wt or dn SHP1 the peak level of EGFR tyrosine phosphorylation induced by EGF, as well as the sensitivity to EGF stimulation, was not altered. However, the overexpression of wt SHP1 led to a slight increase in the kinetics of EGFR dephosphorylation, whereas the overexpression of dn SHP1 led to slightly delayed kinetics of EGFR dephosphorylation. The overexpression of either the wt or dn SHP1 did not lead to any significant increase in Src kinase activity. ^ In NIH3T3 cells, the transient overexpression of SHP1 led to no significant changes in MAP kinase (ERK2) activation by EGF or Akt activation by PDGF. In 3T3H4 cells, the transient overexpression of SHP1 led to no significant changes in MAP kinase (ERK2) activation by heregulin. The transient overexpression of wt SHP1 in the MDA-MB231 cells caused an apparent increase, ranging from 10% to 20%, in the G0/G1 population of the cells with a corresponding decrease in the S phase population. ^ In order to understand the mechanisms by which SHP1 exerts its positive effect on the tumorigenic potential of the MDA-MB231 cells, we employed two-dimensional electrophoresis in an attempt to identify cellular protein(s) with significantly altered tyrosine phosphorylation level upon wt SHP1 overexpression. The overexpression of wt SHP1 but not dn SHP1, leads increased tyrosine phosphorylation of a protein with a molecular weight of approximately 40 kDa and a pI between 5.9 to 6.6. ^
Resumo:
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. ^
Resumo:
Chronic myelogenous leukemia (CML) is characterized cytogenetically by the presence of the Philadelphia chromosome and clinically by the clonal expansion of the hematopoietic stem cells and the accumulation of large numbers of myeloid cells. Philadelphia chromosome results from the reciprocal translocation between chromosomes 9 and 22 [t(9;22)(324;q11)], which fuses parts of the ABL proto-oncogene to 5′ portions of the BCR gene. The product of the fused gene is Bcr-Abl oncoprotein. Bcr-Abl oncoprotein has elevated protein tyrosine kinase activity, and is the cause of Philadelphia chromosome associated leukemias. The Bcr sequence in the fusion protein is crucial for the activation of Abl kinase activity and transforming phenotype of Bcr-Abl oncoprotein. Although the Bcr-Abl oncoprotein has been studied extensively, its normal counterpart, the Bcr protein, has been less studied and its function is not well understood. At this point, Bcr is known to encode a novel serine/threonine protein kinase. In Bcr-Abl positive leukemia cells, we found that the serine kinase activity of Bcr is impaired by tyrosine phosphorylation. Both the Bcr protein sequences within Bcr-Abl and the normal cellular Bcr protein lack serine/threonine kinase activity when they become phosphorylated on tyrosine residues by Bcr-Abl. Therefore, the goal of this study was to investigate the role of Bcr in Bcr-Abl positive leukemia cells. We found that overexpression of Bcr can inhibit Bcr-Abl tyrosine kinase activity, and the inhibition is dependent on its intact serine/threonine kinase function. Using the tet repressible promoter system, we demonstrated that Bcr when induced in Bcr-Abl positive leukemia cells inhibited the Bcr-Abl oncoprotein tyrosine kinase. Furthermore, induction of Bcr also increased the number of cells undergoing apoptosis and inhibited the transforming ability of Bcr-Abl. In contrast to the wild-type Bcr, the kinase-inactive mutant of Bcr (Y328F/Y360F) had no effects on Bcr-Abl tyrosine kinase in cells. Results from other experiments indicated that phosphoserine-containing Bcr sequences within the first exon, which are known to bind to the Abl SH2 domain, are responsible for observed inhibition of the Bcr-Abl tyrosine kinase. Several lines of evidence suggest that the phosphoserine form of Bcr, which binds to the Abl SH2 domain, strongly inhibits the Abl tyrosine kinase domain of Bcr-Abl Previously published findings from our laboratory have also shown that Bcr is phosphorylated on tyrosine residue 177 in Bcr-Abl positive cells and that this form of Bcr recruits the Grb2 adaptor protein, which is known to activate the Ras pathway. These findings implicate Bcr as an effector of Bcr-Abl's oncogenic activity. Therefore based on the findings presented above, we propose a model for dual Function of Bcr in Bcr-Abl positive leukemia cells. Bcr, when active as a serine/threonine kinase and thus autophosphorylating its own serine residues, inhibits Bcr-Abl's oncogenic functions. However, when Ber is tyrosine phosphorylated, its Bcr-Abl inhibitory function is neutralized thus allowing Bcr-Abl to exert its full oncogenic potential. Moreover, tyrosine phosphorylated Bcr would compliment Bcr-Abl's neoplastic effects by the activation of the Ras signaling pathway. ^