Articular cartilage lesions of the knee

We have evaluated the role of various protein kinases on the induction of the gadd (growth arrest and DNA damage inducible) genes, using a panel of protein kinase inhibitors. Our data indicate that three different stress response pathways mediating gadd gene induction are most likely regulated by different protein kinases or combinations of protein kinases. The protein kinase inhibitor staurosporine and the temperature sensitive (ts) p34cdc2 mutant reduced induction by the alkylating agent methylmethane sulfonate (MMS) of the rodent gadd45 and gadd153 genes. However, staurosporine had no effect of the ionizing radiation (IR) induction of the human GADD45. Caffeine and 2-aminopurine, on the other hand, completely blocked this IR induction. Suramin, an antitumor drug that interferes with the interaction of growth factors with their receptors, inhibited the UV radiation induction of GADD45 and GADD153 but had no effect on the MMS and IR pathways. Elevated expression of gadd45 by medium depletion (starvation) was partially reduced by the addition of either genistein or tyrphostin, two protein tyrosine kinase inhibitors, while gadd153 was affected by tyrphostin only. Two inhibitors acting preferentially on cAMP-dependent protein kinase (PKA), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, HCl (H8) and protein kinase inhibitor (PKI), also had a moderate effect on the medium depletion-induced levels of both gadd genes. Thus, these varied effects of inhibitors on gadd gene responses point to important differences in the pathways controlling these responses.     

Abrogation of p53 function affects gadd gene responses to DNA base-damaging agents and starvation

The tumor suppressor p53 is required for induction of its downstream effector genes such as GADD45 and CIP1/WAF1 by ionizing radiation (IR). This response is probably mediated through defined p53 binding sites located in the promoter of CIP1/WAF1 and in the third intron of GADD45. In contrast, the gadd gene stress response to base-damaging agents, such as methylmethane sulfonate (MMS) or UV radiation, or medium depletion (starvation) occurs in all mammalian cells examined to date regardless of p53 status for both GADD45 and also GADD153, which is not IR-responsive in many lines with functional p53. These agents strongly induce the p53 protein and raise the possibility that, although p53 is not required for the typical "gadd" response to these agents, p53 may contribute to these non-IR stress responses. This possibility was confirmed by the finding that disruption of p53 function by transfection with dominant-negative vectors expressing HPV E6, mutant p53, or SV40 T Ag reduced the induction of GADD45 and GADD153 as measured by increases in mRNA and protein levels in human lines with wild-type p53. Similarly, induction of these genes by MMS or UV radiation was consistently stronger in the parental mouse embryo fibroblasts compared to cells derived from mice where both p53 alleles had been deleted. Similar qualitative responses were also seen for CIP1/WAF1. In agreement with reduced induction of p53-regulated genes, the G1 checkpoint activated by MMS or UV radiation was markedly abrogated in p53-wt human MCF-7 breast carcinoma cells by E6 expression. Interestingly, induction of reporter constructs driven by the GADD45 or GADD153 promoters was substantially reduced in human cells transfected with mutant p53 or E6 expression vectors or in cells lacking p53 following treatment with MMS, UV radiation, or starvation. Because neither promoter is inducible by IR, and neither contains a strong p53 binding site, these results indicate that p53 has a synergistic or cooperative role in these non-IR stress responses for both GADD45 and GADD153, and that this role is not mediated through identifiable p53-binding sites.     

Induction of BCL2 family member MCL1 as an early response to DNA damage

When ML-1 human myeloid leukemia cells are exposed to DNA damaging agents, they exhibit dramatic changes in the expression of a variety of gene products. This includes an increase in p53 (wild-type), a decrease in BCL2, a p53-dependent increase in the BCL2 family member BAX, and increases in Growth Arrest and DNA Damage-inducible (GADD) genes such as GADD45; these changes occur as early events in a sequence that culminates in DNA damage-induced apoptosis. DNA damaging agents have now been tested for effects on expression of another BCL2 family member, MCL1, a gene expressed during ML-1 cell differentiation. Expression of MCL1 was found to increase upon exposure of ML-1 cells to various types of DNA damaging agents, including ionizing radiation, ultraviolet radiation, and alkylating drugs. The increase in MCL1 occurred rapidly and was transient, levels of the MCL1 mRNA being elevated within 4 h and having returned to near baseline within 24 h. An increase in the Mcl1 protein was also seen, with the maximal increase occurring at an intermediate dose of IR (5 Gray) and lesser increases occurring at either lower or higher doses. The increase in expression of MCL1 was further studied using a panel of human cell lines that includes cells containing or not containing alterations in p53 as well as cells sensitive or insensitive to the apoptosis-inducing effects of DNA damage. The DNA damage-induced increase in MCL1 mRNA did not depend upon p53 as it was seen in cells lacking functional p53. However, the increase did depend upon susceptibility to apoptosis as it was not seen in cells insensitive to apoptosis-induction by DNA damaging agents. These findings demonstrate that cytotoxic DNA damage causes an increase in the expression of MCL1 along with increases in GADD45 and BAX and a decrease in BCL2. Furthermore, while the increase in GADD45 is seen both in cells that undergo growth arrest and in cells that undergo apoptosis in response to DNA damage, alterations in the profile of expression of BCL2 family members occur exclusively in cells that undergo the apoptotic response, with some family members increasing through p53-dependent (BAX) and others through p53-independent (MCL1) pathways. Overall, expression MCL1 can increase during the induction of cell death as well as during the induction of differentiation.     

c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets

We report here that the expression of virtually all proposed c-Myc target genes is unchanged in cells containing a homozygous null deletion of c-myc. Two noteworthy exceptions are the gene cad, which has reduced log phase expression and serum induction in c-myc null cells, and the growth arrest gene gadd45, which is derepressed by c-myc knockout. Thus, cad and gadd45 are the only proposed targets of c-Myc that may contribute to the dramatic slow growth phenotype of c-myc null cells. Our results demonstrate that a loss-of-function approach is critical for the evaluation of potential c-Myc target genes.     

Regulation of tissue factor gene expression in epithelial cells. Induction by serum and phorbol 12-myristate 13-acetate

Cell-specific expression of tissue factor (TF) in vivo is consistent with its primary role in hemostasis. In addition, TF expression is induced in cultured cells by a variety of agents, including serum and growth factors, which define the TF gene as a "primary response" gene. In this study we examined the signaling pathways and cis-acting regulatory elements required for induction of TF gene expression in HeLa cells in response to serum and the tumor promoter, phorbol 12-myristate 13-acetate (PMA). TF activity and mRNA were induced greater than sixfold in quiescent HeLa cells by serum and PMA. TF mRNA induction by both agonists required intracellular Ca2+ mobilization, whereas inhibition of protein kinase C abolished induction of the TF gene by PMA but had no effect on induction by serum. Functional studies demonstrated that a region of the human TF promoter between -96 and +121 bp contained regulatory elements required for serum and PMA induction. These data indicate that different signaling pathways regulate TF gene expression in response to serum and PMA, although the same cis-acting DNA elements may mediate induction.     

Conversion of a radioresistant phenotype to a more sensitive one by disabling erbB receptor signaling in human cancer cells

Inhibition of cell growth and transformation can be achieved in transformed glial cells by disabling erbB receptor signaling. However, recent evidence indicates that the induction of apoptosis may underlie successful therapy of human cancers. In these studies, we examined whether disabling oncoproteins of the erbB receptor family would sensitize transformed human glial cells to the induction of genomic damage by gamma-irradiation. Radioresistant human glioblastoma cells in which erbB receptor signaling was inhibited exhibited increased growth arrest and apoptosis in response to DNA damage. Apoptosis was observed after radiation in human glioma cells containing either a wild-type or mutated p53 gene product and suggested that both p53-dependent and -independent mechanisms may be responsible for the more radiosensitive phenotype. Because cells exhibiting increased radiation-induced apoptosis were also capable of growth arrest in serum-deprived conditions and in response to DNA damage, apoptotic cell death was not induced simply as a result of impaired growth arrest pathways. Notably, inhibition of erbB signaling was a more potent stimulus for the induction of apoptosis than prolonged serum deprivation. Proximal receptor interactions between erbB receptor members thus influence cell cycle checkpoint pathways activated in response to DNA damage. Disabling erbB receptors may improve the response to gamma-irradiation and other cytotoxic therapies, and this approach suggests that present anticancer strategies could be optimized.     

WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis

The tumor growth suppressor WAF1/CIP1 was recently shown to be induced by p53 and to be a potent inhibitor of cyclin-dependent kinases. In the present studies, we sought to determine the relationship between the expression of WAF1/CIP1 and endogenous regulation of p53 function. WAF1/CIP1 protein was first localized to the nucleus of cells containing wild-type p53 and undergoing G1 arrest. WAF1/CIP1 was induced in wild-type p53-containing cells by exposure to DNA damaging agents, but not in mutant p53-containing cells. The induction of WAF1/CIP1 protein occurred in cells undergoing either p53-associated G1 arrest or apoptosis but not in cells induced to arrest in G1 or to undergo apoptosis through p53-independent mechanisms. DNA damage led to increased levels of WAF1/CIP1 in cyclin E-containing complexes and to an associated decrease in cyclin-dependent kinase activity. These results support the idea that WAF1/CIP1 is a critical downstream effector in the p53-specific pathway of growth control in mammalian cells.     

Adaptation after small bowel resection is attenuated by sialoadenectomy: the role for endogenous epidermal growth factor

Reactive oxygen species generated during the metabolism of the antitumor quinone 3,6-diaziridinyl-1,4-benzoquinone (DZQ) in human colonic carcinoma HCT116 cells lead to the induction of p21 (WAF1, Cip1, or sdi1), an upstream regulator of the retinoblastoma gene product pRb involved G1 cell cycle control. We here demonstrate that the cell cycle was arrested in G2/M phase following supplementation with DZQ of human osteosarcoma Saos-2 cells (lacking both p53 and pRb) and HCT116 cells. DZQ also induced p21 and apoptosis in Saos-2 cells. The transfection of the Rb gene into Saos-2 cells did not alter the level of p21 induction, but changed cell cycle arrest into G1 phase and prevented apoptosis. These findings suggest that quinones may lead to a p53-independent and pRb-preventable G2/M arrest and apoptosis, which correlate with p21 induction.