[Frontiers in Bioscience 2, a37-45, November 1, 1997]

	[Frontiers in Bioscience 2, a37-45, November 1, 1997] Reprints PubMed CAVEAT LECTOR
	A p53 GROWTH ARREST PROTECTS FIBROBLASTS FROM ANTICANCER AGENTS E. Siobhan McCormack, Arthur M. Bruskin, Gary V. Borzillo OSI Pharmaceuticals Inc., 106 Charles Lindbergh Blvd., Uniondale, NY 11553-3649 Received 10/27/97 Accepted October 31, 1997 5. DISCUSSION The G1 phase of the cell cycle is a period in which cells can reversibly arrest in response to various environmental cues and the activation of cell cycle checkpoints. The arresting pathways do not necessarily utilize identical signaling mechanisms, but appear to share the ability to inhibit cyclin-dependent kinases (such as CDK4) that operate in G1 (reviewed in 17). We had previously reported that a TGFB3 growth arrest could protect epithelial and hematopoietic cells from anticancer drugs in vitro and in vivo, and to reduce the severity of oral mucositis induced by chemotherapy in an animal model (1, 2). In tissue culture, preincubation of CCL64 epithelial cells with TGFB3 improved survival following exposure to agents (vinblastine, etoposide, taxol, Ara-c) which act predominantly in S or M phase (5). However, the effects of TGFB3 were reduced for the drugs cisplatin and doxorubicin, which are toxic to cells throughout the cell cycle. In the present study, we examined a different growth arrest mechanism (p53) for the chemoprotection of cells exposed to anticancer drugs. We anticipated from previous studies (18, 19) that p53 (or other arrest mechanisms) might differ from the TGFB family regarding the profile of drugs where protection was observed. Here, we report that ts p53 mutations behaved similarly to TGFB: improving survival from drugs which target DNA synthesis or mitosis selectively, but showing little or no effect against drugs active throughout the cell cycle. The p53 protein functions as part of a cell cycle checkpoint that can be activated in response to DNA damage. Whereas untransformed fibroblasts and many adherent cell lines undergo growth arrest following p53 activation, other cells are triggered into undergoing apoptosis (20-22). Examples include subsets of murine hematopoietic and gastrointestinal cells exposed to ionizing radiation, or cells lacking normal Rb function due to the ectopic expression of E2F or presence of viral oncoproteins. In such cell types, wild-type p53 status has a negative effect on survival, and apoptosis is further increased after drug or radiation treatment. Thus, since one function of p53 is to delete damaged cells from an organism, the choice of cell type is critical for examining any positive effects of p53 on survival. Several groups have previously analyzed the differences between the p53^(+/+)and p53^(-/-) genotype in a cell background where p53 activation leads to growth arrest. Unlike the present study, where we pre-arrested cells for chemoprotection, these earlier reports examined the effect of anticancer drugs on dividing cultures. Hawkins and coworkers (18) reported that human foreskin fibroblasts (HFF) were more resistant to killing by cisplatin, carboplatin and paclitaxel than derivative lines in which p53 function was abrogated by the human papillomavirus (HPV) E6 protein. Fan and coworkers (19) reported that both MCF-7 and RKO cells were more resistant to cisplatin than derivative lines overexpressing either HPV E6 or p53 mutations. Moreover, abrogation of a checkpoint operative in G2 with the caffeine derivative pentoxfylline led to a further differential killing of the p53 defective cells. Taken together with the present study, we would argue that novel mechanisms for p53 activation could be expoited to promote the survival of some lineages exposed to many drugs or radiation. Our inability to protect cells from cisplatin with either p53 or TGFB3 (5) probably relates to the ability of this drug to kill effectively cells in G1 (16), where our pre-arrested cells were synchronized. In addition, our G1-arrested cells do not benefit from the contribution made by p53 to checkpoints in S or G2 phases (23). A complication of engaging the p53 pathway to chemoprotect the appropriate cells is that p53 is physiologically activated by cellular insults (DNA damage, virus infection, hypoxia). Safer mechanisms of activation would be needed to exploit the p53 growth arrest. The specific DNA binding function of wt p53 is negatively regulated by sequences in its carboxy terminus, which can be truncated to constitutively activate DNA binding (24). Peptides based on c-terminal sequences have been shown to activate p53 for specific DNA binding in vitro and in vivo (25). Selivanova and coworkers (26) reported that a peptide corresponding to residues 361-382 could activate DNA binding and transcription activation of wt (and some mt) forms of p53. When the peptide was engineered to translocate cell membranes, p53 transactivation increased and the growth of tumor lines was inhibited in a p53-dependent fashion. Thus, an emerging question is whether small molecular weight drugs will be identified which restore normal functions to some p53 mutations, and whether the same agents (or the p53 peptides themselves) would protect normal dividing cells from DNA damaging agents.

[Frontiers in Bioscience 2, a37-45, November 1, 1997]
Reprints
PubMed
CAVEAT LECTOR

A p53 GROWTH ARREST PROTECTS FIBROBLASTS FROM ANTICANCER AGENTS

E. Siobhan McCormack, Arthur M. Bruskin, Gary V. Borzillo

OSI Pharmaceuticals Inc., 106 Charles Lindbergh Blvd., Uniondale, NY 11553-3649

Received 10/27/97 Accepted October 31, 1997

5. DISCUSSION

The G1 phase of the cell cycle is a period in which cells can reversibly arrest in response to various environmental cues and the activation of cell cycle checkpoints. The arresting pathways do not necessarily utilize identical signaling mechanisms, but appear to share the ability to inhibit cyclin-dependent kinases (such as CDK4) that operate in G1 (reviewed in 17). We had previously reported that a TGFB3 growth arrest could protect epithelial and hematopoietic cells from anticancer drugs in vitro and in vivo, and to reduce the severity of oral mucositis induced by chemotherapy in an animal model (1, 2). In tissue culture, preincubation of CCL64 epithelial cells with TGFB3 improved survival following exposure to agents (vinblastine, etoposide, taxol, Ara-c) which act predominantly in S or M phase (5). However, the effects of TGFB3 were reduced for the drugs cisplatin and doxorubicin, which are toxic to cells throughout the cell cycle. In the present study, we examined a different growth arrest mechanism (p53) for the chemoprotection of cells exposed to anticancer drugs. We anticipated from previous studies (18, 19) that p53 (or other arrest mechanisms) might differ from the TGFB family regarding the profile of drugs where protection was observed. Here, we report that ts p53 mutations behaved similarly to TGFB: improving survival from drugs which target DNA synthesis or mitosis selectively, but showing little or no effect against drugs active throughout the cell cycle.

The p53 protein functions as part of a cell cycle checkpoint that can be activated in response to DNA damage. Whereas untransformed fibroblasts and many adherent cell lines undergo growth arrest following p53 activation, other cells are triggered into undergoing apoptosis (20-22). Examples include subsets of murine hematopoietic and gastrointestinal cells exposed to ionizing radiation, or cells lacking normal Rb function due to the ectopic expression of E2F or presence of viral oncoproteins. In such cell types, wild-type p53 status has a negative effect on survival, and apoptosis is further increased after drug or radiation treatment. Thus, since one function of p53 is to delete damaged cells from an organism, the choice of cell type is critical for examining any positive effects of p53 on survival. Several groups have previously analyzed the differences between the p53^(+/+)and p53^(-/-) genotype in a cell background where p53 activation leads to growth arrest. Unlike the present study, where we pre-arrested cells for chemoprotection, these earlier reports examined the effect of anticancer drugs on dividing cultures. Hawkins and coworkers (18) reported that human foreskin fibroblasts (HFF) were more resistant to killing by cisplatin, carboplatin and paclitaxel than derivative lines in which p53 function was abrogated by the human papillomavirus (HPV) E6 protein. Fan and coworkers (19) reported that both MCF-7 and RKO cells were more resistant to cisplatin than derivative lines overexpressing either HPV E6 or p53 mutations. Moreover, abrogation of a checkpoint operative in G2 with the caffeine derivative pentoxfylline led to a further differential killing of the p53 defective cells. Taken together with the present study, we would argue that novel mechanisms for p53 activation could be expoited to promote the survival of some lineages exposed to many drugs or radiation. Our inability to protect cells from cisplatin with either p53 or TGFB3 (5) probably relates to the ability of this drug to kill effectively cells in G1 (16), where our pre-arrested cells were synchronized. In addition, our G1-arrested cells do not benefit from the contribution made by p53 to checkpoints in S or G2 phases (23).

A complication of engaging the p53 pathway to chemoprotect the appropriate cells is that p53 is physiologically activated by cellular insults (DNA damage, virus infection, hypoxia). Safer mechanisms of activation would be needed to exploit the p53 growth arrest. The specific DNA binding function of wt p53 is negatively regulated by sequences in its carboxy terminus, which can be truncated to constitutively activate DNA binding (24). Peptides based on c-terminal sequences have been shown to activate p53 for specific DNA binding in vitro and in vivo (25). Selivanova and coworkers (26) reported that a peptide corresponding to residues 361-382 could activate DNA binding and transcription activation of wt (and some mt) forms of p53. When the peptide was engineered to translocate cell membranes, p53 transactivation increased and the growth of tumor lines was inhibited in a p53-dependent fashion. Thus, an emerging question is whether small molecular weight drugs will be identified which restore normal functions to some p53 mutations, and whether the same agents (or the p53 peptides themselves) would protect normal dividing cells from DNA damaging agents.