Purpose: Bendamustine has shown clinical activity in patients with disease refractory to conventional alkylator chemotherapy. The purpose of this study was to characterize the mechanisms of action of bendamustine and to compare it with structurally related compounds. Experimental Design: Bendamustine was profiled in the National Cancer Institute in vitro antitumor screen. Microarray-based gene expression profiling, real-time PCR, immunoblot, cell cycle, and functional DNA damage repair analyses were used to characterize response to bendamustine and compare it with chlorambucil and phosphoramide mustard. Results: Bendamustine displays a distinct pattern of activity unrelated to other DNA-alkylating agents. Its mechanisms of action include activation of DNA-damage stress response and apoptosis, inhibition of mitotic checkpoints, and induction of mitotic catastrophe. In addition, unlike other alkylators, bendamustine activates a base excision DNA repair pathway rather than an alkyltransferase DNA repair mechanism. Conclusion: These results suggest that bendamustine possesses mechanistic features that differentiate it from other alkylating agents and may contribute to its distinct clinical efficacy profile.

We have examined several types of tumor cell lines and shown that they invariably expressed little or no Egr-1, in contrast to their normal counterparts. We have previously shown that the expression of exogenous Egr-1 in human breast and other tumor cells markedly reduces transformed growth and tumorigenicity. We therefore hypothesized that the loss of Egr-1 expression plays a role in transformation. All human and mouse breast cancer cell lines and tumors examined had reduced Egr-1 expression compared with their normal counterparts. Reduced Egr-1 expression was also observed in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, and this level increased to normal levels in tumors that regressed after tamoxifen treatment. We concluded, therefore, that loss of Egr-1 expression may play a role in the deregulation of normal growth in the tumorigenic process and that Egr-1 acts as a tumor suppressor gene. The early growth response gene, Egr-1 (Sukhatme et al., 1988) (zfp-6 in Standardized Genetic Nomenclature for Mice), also known as NGF1A, Krox 24, zif268 and T1S-8, encodes a protein with 3 adjacent zinc-finger motifs, structures that are present in many DNA-binding transcription factors. The Egr family of proteins consists of 4 members that all bind to the same DNA element: GCGGGGGCG or GCGT/GGGGCG (Christy and Nathans, 1989;LeMaire et al., 1988) because of the remarkable conservation of their zinc-finger DNA binding domains. The Egr family is a highly evolutionarily conserved set of genes but it has proved difficult to define a precise role. One member of the family, WT1, has a homologous zinc-finger domain with 4 fingers that bind to the same DNA motif. WT1 has been categorized as a tumor suppressor gene that is mutant in Wilms' tumor disease in children (Rauscher, 1993).We have shown that Egr-1 has tumor suppressor properties and that the DNA-binding domain is necessary for this activity (Huang et al., 1994a;. The over-expression of Egr-1 in transformed cells suppressed their growth in soft agar and their growth as tumors in athymic mice. In contrast, further inhibition of Egr-1 in mouse transformed cells using antisense-expression vectors increased the transformed character of the cells (Huang et al., 1994b). During the analysis of a range of tumor cell lines, we observed that the expression of Egr-1 was often anomalously low. We have shown that the over-expression of Egr-1 can restore normal growth patterns to these tumor cells, which suggested that the loss of this transcription factor might be either a cause of, or may accompany, the loss of growth control leading to tumor production. Some of this growth down-regulation is due to the induction of Transforming Growth Factor-b expression (Liu et al., 1996). This factor is a growth inhibitor of epithelial cells that express the receptor and accounts for reduced growth rates in some cell types.We have examined here the expression of Egr-1 in human breast tissues and mammary cell lines and tissues. The data strongly support the hypothesis that Egr-1...

We have examined several types of tumor cell lines and shown that they invariably expressed little or no Egr-1, in contrast to their normal counterparts. We have previously shown that the expression of exogenous Egr-1 in human breast and other tumor cells markedly reduces transformed growth and tumorigenicity. We therefore hypothesized that the loss of Egr-1 expression plays a role in transformation. All human and mouse breast cancer cell lines and tumors examined had reduced Egr-1 expression compared with their normal counterparts. Reduced Egr-1 expression was also observed in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, and this level increased to normal levels in tumors that regressed after tamoxifen treatment. We concluded, therefore, that loss of Egr-1 expression may play a role in the deregulation of normal growth in the tumorigenic process and that Egr-1 acts as a tumor suppressor gene. The early growth response gene, Egr-1 (Sukhatme et al., 1988) (zfp-6 in Standardized Genetic Nomenclature for Mice), also known as NGF1A, Krox 24, zif268 and T1S-8, encodes a protein with 3 adjacent zinc-finger motifs, structures that are present in many DNA-binding transcription factors. The Egr family of proteins consists of 4 members that all bind to the same DNA element: GCGGGGGCG or GCGT/GGGGCG (Christy and Nathans, 1989;LeMaire et al., 1988) because of the remarkable conservation of their zinc-finger DNA binding domains. The Egr family is a highly evolutionarily conserved set of genes but it has proved difficult to define a precise role. One member of the family, WT1, has a homologous zinc-finger domain with 4 fingers that bind to the same DNA motif. WT1 has been categorized as a tumor suppressor gene that is mutant in Wilms' tumor disease in children (Rauscher, 1993).We have shown that Egr-1 has tumor suppressor properties and that the DNA-binding domain is necessary for this activity (Huang et al., 1994a;. The over-expression of Egr-1 in transformed cells suppressed their growth in soft agar and their growth as tumors in athymic mice. In contrast, further inhibition of Egr-1 in mouse transformed cells using antisense-expression vectors increased the transformed character of the cells (Huang et al., 1994b). During the analysis of a range of tumor cell lines, we observed that the expression of Egr-1 was often anomalously low. We have shown that the over-expression of Egr-1 can restore normal growth patterns to these tumor cells, which suggested that the loss of this transcription factor might be either a cause of, or may accompany, the loss of growth control leading to tumor production. Some of this growth down-regulation is due to the induction of Transforming Growth Factor-b expression (Liu et al., 1996). This factor is a growth inhibitor of epithelial cells that express the receptor and accounts for reduced growth rates in some cell types.We have examined here the expression of Egr-1 in human breast tissues and mammary cell lines and tissues. The data strongly support the hypothesis that Egr-1 ...

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