Biomedical subjects

Brigitte M Pützer

Publications and source records attributed to Brigitte M Pützer.

10 recordsLinked to original sources

PubMed2004

Alterations of DeltaTA-p 73 splice transcripts during melanoma development and progression.

In the last 2 years, it has become apparent that the p53-family members p53 and p73 play fundamentally different roles in human malignancies. In contrast to p53, many studies on cancer patients failed to detect mutational inactivation of p73 and reported overexpression of wild-type p73 instead. A possible explanation was provided by the recent discovery of N-terminal truncated isoforms of p73 (DeltaTA-p73) that act as dominant-negative inhibitors of wild-type p53 and TA-p73 and result in tumor growth in nude mice. We investigated the role of DeltaTA-p73 in the development and progression of human melanomas, which lack p53 mutations. We analyzed 8 benign melanocytic nevi, 8 primary melanomas and 19 melanoma metastases for alterations of TA-p73 and DeltaTA-p73 expression using isoform-specific real-time RT-PCR. Based on our results, p73Deltaex2 and Deltaex2/3 spliced transcripts derived from the first promoter were significantly up-regulated in melanoma metastases, whereas DeltaN-p73 generated from the second promoter was the predominant isoform in benign nevi. Moreover, increased expression of p73Deltaex2 and p73Deltaex2/3 correlated with high-levels of both TA-p73 and E2F1. Our data suggest a potential function of DeltaTA-p73 splice isoforms in melanoma progression.

DNA-Binding Proteins↗

PubMed2003

Mechanism of E2F1-induced apoptosis in primary vascular smooth muscle cells.

OBJECTIVE: The transcription factor E2F1 serves as a major regulator of the cell-cycle by controlling G1-S phase transition. However, apart from its proliferative function high levels of deregulated E2F1 are capable of inducing apoptosis depending on the cellular context. In particular the tumor suppressor p53 and its homologue p73 are implicated in this proapoptotic function. METHODS: Here, we investigated the mechanistic basis for E2F1-mediated apoptosis in vascular smooth muscle cells (VSMCs) which have previously been shown to be E2F1-responsive. RESULTS: Interestingly, E2F1-expression in these cells induced clear signs of apoptosis in the absence of any proliferative activity. Although cell-cycle regulated genes such as CCNE1 and CDC25A were activated, BrdU-staining revealed no S-phase entry. Instead, a rapid loss of cell viability by induction of apoptosis was observed. Using a transactivation-defective E2F1-mutant, we show that apoptosis induction is independent of the transactivation function and therefore independent of ARF and p73. However, this mutant retains its ability to stabilize and phosphorylate p53, suggesting that p53 is sufficient for the effect of E2F1. CONCLUSION: VSMCs therefore represent a cellular system in which the transactivation-independent, proapoptotic activity of E2F1 is the primary cellular function. Ectopic expression of E2F1 might therefore be a suitable therapy to prevent VSMC hyperproliferation.

Adenoviridae↗

PubMed2003

Gene therapeutic approaches for medullary thyroid carcinoma treatment.

Medullary thyroid carcinoma (MTC), a neoplasm of thyroid C-cells, is characterized by dominant activating mutations in the RET proto-oncogene. Currently therapy is restricted to surgical removal of all neoplastic tissue lacking alternative forms of treatment such as chemotherapy or radiotherapy. Therefore MTC is a particularly attractive target for gene therapeutic approaches. Many promising gene therapy strategies have been used in various animal models of MTC, showing enhanced antitumoral efficacy, and these will hopefully extend our current standard of care in the future. These approaches can tentatively be subdivided into four groups: (a) Inhibition of oncogenic RET signaling, (b) suicide gene therapy, (c) immunotherapy, and (d) combination of immunotherapy and suicide approaches. To block oncogenic signal transduction dominant-negative RET mutants were delivered into tumor cells and found to possess strong antineoplastic activity, including tumor growth suppression and increased animal survival. Suicide gene therapeutic approaches applied to MTC treatment featured either gene transfer of herpes simplex virus thymidine kinase with concomitant application of ganciclovir or delivery of nitric oxide synthase II. Here antitumor effects were attributed to the occurrence of substantial bystander activities. Immunotherapy approaches comprised stimulation of immune response by delivery of interleukin 2 or 12. Finally, treatment with herpes simplex virus thymidine kinase/ganciclovir in combination with interleukin 2 was found to be superior over either treatment alone. This review discusses the various gene therapeutic approaches applied to MTC treatment in detail, gives an overview on the diverse vector systems used to achieve efficient transduction of thyroid cancer cells, and points out the strategies employed to accomplish target cell selective gene expression thereby contributing to enhanced safety of gene therapy for MTC

Animals↗

PubMed2003

p73 is effective in p53-null pancreatic cancer cells resistant to wild-type TP53 gene replacement.

Novel therapies such as gene therapy are needed for the treatment of pancreatic carcinomas. Here we show that adenovirus-mediated p73 overexpression results in a strong induction of apoptosis, whereas the effect of p53 varies between different cell lines. In particular, p53-negative AsPC-1 cells are resistant to p53-mediated apoptosis. In these cells, only ectopically expressed p73 activates the proapoptotic p53 target P53AIP1, whereas phosphorylation of p53 at Ser-46, shown to regulate transcriptional activation of P53AIP1, is missing. Our findings support the use of p73 as an anticancer drug in p53-null pancreatic cancer cells that are resistant to wild-type TP53 gene replacement.

Adenocarcinoma↗

PubMed2003

Inactivation of retinoblastoma (RB) tumor suppressor by oncogenic isoforms of the p53 family member p73.

The p53 family includes three members that share significant sequence homology, yet exhibit fundamentally different functions in tumorigenesis. Whereas p53 displays all characteristics of a classical tumor suppressor, its homologues p63 and p73 do not. We have previously shown, that NH(2)-terminally truncated isoforms of p73 (Delta TA-p73), which act as dominant-negative inhibitors of p53 are frequently overexpressed in cancer cells. Here we provide evidence that Delta TA-p73 isoforms also affect the retinoblastoma protein (RB) tumor suppressor pathway independent of p53. Delta TA-p73 isoforms inactivate RB by increased phosphorylation, resulting in enhanced E2F activity and proliferation of fibroblasts. By inactivating the two major tumor suppressor pathways in human cells they act functionally analogous to several viral oncoproteins. These findings provide an explanation for the fundamentally different functions of p53 and p73 in tumorigenesis.

Adenoviridae↗

PubMed2002

Transactivation-deficient DeltaTA-p73 acts as an oncogene.

The recently discovered p53-family member p73 displays significant homology to p53, but data from primary tumors and knockout mice argue against p73 being a classical tumor suppressor. We report on the overexpression of NH(2)-terminally truncated, transactivation-deficient p73 proteins (DeltaTA-p73) in human cancer cells. Moreover, we show that DeltaTA-p73 overexpression results in malignant transformation of NIH3T3 fibroblasts and tumor growth in nude mice, thereby providing the experimental evidence for an oncogenic function of DeltaTA-p73. Apparently, increased expression of NH(2)-terminally truncated p73 isoforms conveys the TP73 gene with oncogenic activity that appears to be actively selected for during tumor development.

3T3 Cells↗

PubMed2002

Gene expression changes in response to E2F1 activation.

The p16/RB/E2F regulatory pathway, which controls transit through the G1 restriction point of the cell cycle, is one of the most frequent targets of genetic alterations in human cancer. Any of these alterations results in the deregulated expression of the transcription factor E2F, one of the key mediators of cell cycle progression. Under these conditions, E2F1 also participates in the induction of apoptosis by a p53-dependent pathway, and independently of p53. Recently, we identified the p53-homolog p73 as a first direct target of p53-independent apoptosis. Here, we used a cDNA microarray to screen an inducible E2F1-expressing Saos-2 cell line for E2F1 target genes. Expression analysis by cDNA microarray and RT-PCR revealed novel E2F1 target genes involved in E2F1-regulated cellular functions such as cell cycle control, DNA replication and apoptosis. In addition, the identification of novel E2F1 target genes participating in the processes of angiogenesis, invasion and metastasis supports the view that E2F1 plays a central role in many aspects of cancer development. These results provide new insight into the role of E2F1 in tumorigenesis as a basis for the development of novel anti-cancer therapeutics.

Cell Cycle Proteins↗

PubMed2002

Transactivation-deficient Delta TA-p73 inhibits p53 by direct competition for DNA binding: implications for tumorigenesis.

The p53 family member p73 displays significant structural and functional homology to p53. However, instead of mutational inactivation, overexpression of wild-type p73 has been reported in various tumor types compared with normal tissues, arguing against a classical tumor suppressor function. Recently, N-terminally truncated, transactivation-deficient p73 isoforms (DeltaTA-p73) have been identified as a second class of p73 proteins. Because overexpression of p73 in tumors includes DeltaTA-p73, we further characterized these novel p73 isoforms. We show that DeltaTA-p73 retains DNA-binding competence but lacks transactivation functions, resulting in an inability to induce growth arrest and apoptosis. Importantly, DeltaTA-p73 acts as a dominant-negative inhibitor of p53 and full-length p73 (TA-p73). We demonstrate that inhibition of p53 involves competition for DNA binding, whereas TA-p73 can be inhibited by direct protein-protein interaction. Further, we show that up-regulation of endogenous p73 just like ectopic overexpression of DeltaTA-p73 confers resistance to p53-mediated apoptosis induced by the chemotherapeutic agent H-7. Because inhibition of p53 is a common theme in human cancer, our data strongly support a role of DeltaTA-p73 expression for tumor formation.

Adenoviridae↗

PubMed2002

Improved treatment of pancreatic cancer by IL-12 and B7.1 costimulation: antitumor efficacy and immunoregulation in a nonimmunogenic tumor model.

Ductal pancreatic adenocarcinoma is one of the commonest and most lethal cancers in the Western world. Unfortunately, recent advances in diagnostics, staging, and therapy in pancreatic carcinoma have not resulted in significant improvements in long-term survival. We have previously shown that adenovirus (Ad)-mediated coexpression of interleukin-12 (IL-12) and the costimulatory molecule B7.1 is extremely efficient in inducing regression of highly immunogenic transplanted and nontransplanted tumors. Here, we examined the antitumor efficacy of IL-12- and B7.1-based immunotherapy against a nonimmunogenic murine model of ductal pancreatic cancer. Compared with AdIL-12 treatment alone, single intratumoral injection of AdIL-12/B7.1 led to a prolonged immune response and mediated complete regression in 80% of treated animals. After rechallenge with parental tumor cells, 70% of cured mice remained tumor-free, suggesting that protective immunity had been induced. The antitumoral response was associated with upregulation of H-2K(b) and Abcb2 expression, whereas other components of the proteasome (Abcb3, Psmb9, and Psmb8) were not affected. These data indicate that upregulation of the antigen presentation machinery by AdIL-12/B7.1 may be a therapeutic rationale for nonimmunogenic, therapy-resistant pancreatic cancer.

Animals↗

PubMed2002

A new therapeutic approach in medullary thyroid cancer treatment: inhibition of oncogenic RET signaling by adenoviral vector-mediated expression of a dominant-negative RET mutant.

BACKGROUND: Mutations in the RET proto-oncogene that result in constitutive tyrosine kinase activity are the underlying cause for the development of medullary thyroid cancer (MTC). To investigate an alternative strategy in MTC treatment, we took advantage of a dominant-negative RET (dn-RET) mutant, Ret(51)HSCR32, which inhibits oncogenic signal transduction by retaining the oncogenic RET protein in the endoplasmic reticulum, thereby reducing the amount of oncogenic RET protein from the cell surface. METHODS; We constructed an adenoviral (Ad) vector expressing dn-RET under control of the C-cell specific synthetic calcitonin promoter TSE2.CP1 (AdTSE2.CP1-RET(51)HSCR32) and investigated the effect of dn-RET on cell growth of MTC-derived TT cells. RESULTS: Analysis of the subcellular localization of endogenous oncogenic RET protein showed a significant dominant-negative effect of Ad vector-delivered dn-RET in TT cells, resulting in a strong inhibition of cell viability. The observed effect is partially dependent on growth inhibition and possibly apoptosis induction. CONCLUSIONS: In the present study, growth of human MTC cells was successfully inhibited by Ad vector-mediated delivery of RET(51)HSCR32, suggesting that inhibition of oncogenic RET receptor tyrosine kinase expression by a dn-RET mutant might be a powerful approach for in vivo therapy of MTC.

Adenoviridae↗