p53 and SCFFbw7 cooperatively restrain cyclin E-associated genome instability.

Cancers often exhibit high levels of cyclin E expression, and aberrant cyclin E activity causes genomic instability and increased tumorigenesis. Two tumor suppressor pathways protect cells against cyclin E deregulation. The p53 pathway is induced by excess cyclin E in primary cells and opposes cyclin E activity through induction of p21Cip1. In contrast, the Fbw7 pathway targets cyclin E for degradation, and Fbw7 mutations occur commonly in cancers. We investigated the cooperativity of these two pathways in countering cyclin E-induced genomic instability in primary human cells. We find that loss of p53 and Fbw7 synergistically unmasks cyclin E-induced instability. In normal cells, impaired cyclin E degradation produces genome instability, but this is rapidly mitigated by induction of p53 and p21. In contrast, p53 loss allows the high level of cyclin E kinase activity that results from Fbw7 loss to persist and continuously drive genome instability. Moreover, p21 plays a critical role in suppressing cyclin E when Fbw7 is disabled, and in the absence of p21, sustained cyclin E activity induces rapid cell death via apoptosis. These data directly demonstrate the cooperative roles of these Fbw7 and p53 pathways in restraining cyclin E activity and its associated genome instability.

Intracellular expression of the anti-erbB-2 sFv N29 fails to accomplish efficient target modulation.

The use of intracellular single chain antibodies has recently emerged as a highly efficient method of down-regulating or ablating protein expression. In this regard, we have demonstrated that a single chain antibody directed against the extracellular domain of the erbB-2 molecule causes a specific toxicity in erbB-2 positive tumor types. To further investigate the mechanism of this effect, we developed a second anti-erbB-2 sFv predicted to recognize an alternate extracellular epitope of the erbB-2 molecule. When produced as a secreted protein from the erbB-2 negative COS-1 cell line, this sFv binds specifically to erbB-2 positive cells, indicating that cellular machinery is able to produce a properly folded and functional sFv protein. However, by several assays, this sFv was shown to be unable to retain the erbB-2 protein within the ER. These negative results have implications for the evaluation and utilization of sFv knockout strategies in experimental contexts.

Targeted eradication of ovarian cancer mediated by intracellular expression of anti-erbB-2 single-chain antibody.

OBJECTIVE: Overexpression of the tyrosine kinase receptor erbB-2 is important in the pathogenesis of a variety of neoplasms including ovarian cancer. As a strategy to selectively eradicate erbB-2-overexpressing tumor cells, an anti-erbB-2 single-chain immunoglobin (sFv) gene was constructed to direct expression of intracellular anti-erbB-2 antibody. The purpose of this study is to establish the antitumorigenicity of this strategy in in vitro and in vivo models. METHODS: An anti-erbB-2 sFv construct containing an endoplasmic reticulum (ER)-directed leader sequence was transiently expressed in the human ovarian carcinoma cell line SKOV3 using the adenovirus-polylysine vector. SKOV3 cells transfected with a non-ER form of an anti-erbB-2 sFv construct or an irrelevant plasmid DNA served as controls. Antitumorigenicity, as measured by anchorage-independent growth in soft agar and subcutaneous (sc) tumor formation, was analyzed. The ability to achieve a biological effect with relevant sFv in murine orthotopic xenograft models and in primary human ovarian cancer cells was also evaluated. RESULTS: The ER form of anti-erbB-2 sFv was found to exert a marked antineoplastic effect on the SKOV3 cell line resulting in an arrest of anchorage-independent growth. A significant increase in sc tumor volume was noted in animals challenged with control constructs. In marked contrast, complete tumor eradication was noted at necropsy 80 days after sc transplantation in the group challenged with the ER-directed anti-erbB-2 sFv gene. Intraperitoneal treatment of malignant ascites in human tumor xenograft models with the ER form of the anti-erbB-2 sFv gene resulted in profound downregulation of cell surface erbB-2 in retrieved ovarian cancer cells. The ER-directed anti-erbB-2 sFv also elicited a significant cytotoxic effect in transfected primary ovarian cancer cells obtained from a patient with malignant ascites. CONCLUSION: The ability to selectively "knock out" erbB-2 demonstrates that this strategy can induce a significant antineoplastic effect in ovarian cancer cells overexpressing this growth factor receptor. In addition, the ability to accomplish selective abrogation of erbB-2 expression in animal treatment models and to transfect and eradicate primary ovarian cancer cells justifies further investigation of this novel strategy in ovarian cancer patients.