IMR-90 Normal Human Fibroblasts Research Articles

Actin Dysfunction Induces Cell Cycle Delay at G2/M with Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts.

The actin cytoskeleton plays a key role in the entry of mitosis as well as in cytokinesis. In a previous study, we showed that actin disruption delays mitotic entry at G2/M by sustained activation of extracellular signal-related kinase 1/2 (ERK1/2) in primary cells but not in transformed cancer cell lines. Here, we examined the mechanism of cell cycle delay at G2/M by actin dysfunction in IMR-90 normal human fibroblasts. We observed that de-polymerization of actin with cytochalasin D (CD) constitutively activated ribosomal S6 kinase (RSK) and induced inhibitory phosphorylation of Cdc2 (Tyr 15) in IMR-90 cells. In the presence of an actin defect in IMR-90 cells, activating phosphorylation of Wee1 kinase (Ser 642) and inhibitory phosphorylation of Cdc25C (Ser 216) was also maintained. However, when kinase-dead RSK (DN-RSK) was over-expressed, we observed sustained activation of ERK1/2, but no delay in the G2/M transition, demonstrating that RSK functions downstream of ERK in cell cycle delay by actin dysfunction. In DN-RSK overexpressing IMR-90 cells treated with CD, phosphorylation of Cdc25C (Ser 216) was blocked and phosphorylation of Cdc2 (Tyr 15) was decreased, but the phosphorylation of Wee1 (Ser 642) was maintained, demonstrating that RSK directly controls phosphorylation of Cdc25C (Ser 216), but not the activity of Wee1. These results strongly suggest that actin dysfunction in primary cells activates ERK1/2 to inhibit Cdc2, delaying the cell cycle at G2/M by activating downstream RSK, which phosphorylates and blocks Cdc25C, and by directly activating Wee1.

Stathmin/Op18 depletion induces genomic instability and leads to premature senescence in human normal fibroblasts.

Stathmin/oncoprotein18 regulates microtubule dynamics and participates in mitotic entry and exit. We isolated stathmin as a physically interacting partner of KIFC1, a minus-end-directed kinesin functioning in bipolar spindle formation and maintenance. We found that stathmin depletion leads to multipolar spindle formation in IMR-90 normal human fibroblasts. Stathmin-depleted IMR-90 cells showed early mitotic delay but managed to undergo chromosome segregation by forming multiple poles or pseudo-bipoles. Consistent with these observations, lagging chromosomes, and micronuclei were elevated in stathmin-depleted IMR-90 cells, demonstrating that stathmin is essential for maintaining genomic stability during mitosis in human cells. Genomic instability induced by stathmin depletion led to premature senescence without any indication of cell death in normal IMR-90 cells. Double knock-down of both stathmin and p53 also did not induce cell death in IMR-90 cells, while the stathmin knock-down triggered apoptosis in p53-proficient human lung adenocarcinoma cells. Our results suggest that stathmin is essential in bipolar spindle formation to maintain genomic stability during mitosis, and the depletion of stathmin prevents the initiation of chromosome instability by inducing senescence in human normal fibroblasts.

Pretreatment of human colon tumor cells with DNA methylating agents inhibits their ability to repair chloroethyl monoadducts.

We have recently shown that a variety of human tumor cell lines are capable of preventing chloroethylnitrosourea (CNU)-induced DNA crosslinks, presumably by removing guanine O6-chloroethyl DNA monoadducts before crosslinks can form. Those cells capable of preventing crosslinking were of the Mer+ (Methylation repair) phenotype, and have been shown to be proficient at repair of O6-methyl-guanine adducts by the repair enzyme guanine-O6-methyl-transferase. Mer- tumor cell lines are: deficient at O6-methyl-guanine repair, incapable of preventing CNU interstrand crosslinking, and have recently been shown to lack the repair enzyme O6-methyl-transferase. We wish to report that pretreatment of Mer+ cells (HT-29 human colon carcinoma cells and IMR-90 normal human fibroblasts) with the DNA methylating agent MNNG, under conditions which should inactivate O6-methyl-transferase, apparently saturates the monoadduct repair system, and allows CNU to form interstrand crosslinks in these cells. This effect was also seen when MNU pretreatment was used, but not with MMS or streptozotocin. The formation of CNU-induced interstrand crosslinks following MNNG or MNU pretreatment was coincident with a dramatic increase in cytotoxicity as measured by colony formation assays. In contrast, cytotoxicity was only slightly increased when MMS or streptozotocin pretreatment was used.

Pretreatment of normal human fibroblasts and human colon carcinoma cells with MNNG allows chloroethylnitrosourea to produce DNA interstrand crosslinks not observed in cells treated with chloroethylnitrosourea alone.

Chloroethylnitrosoureas (CNU) are antitumor agents which produce DNA interstrand crosslinks. We have proposed that crosslinks are produced in DNA via monoadduct formation at the guanine-O6 position, followed by a delayed reaction with the opposite DNA strand. Human cells are known to differ in their capacity to repair the O6-methylguanine lesion. One example of this repair capacity is the ability of cells to reactivate adenovirus which has been damaged by in vitro treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Cells that repair the virus are designated Mer+ and deficient cells Mer-. In a recent report, we showed a clear correlation between CNU-induced DNA interstrand crosslinking and the Mer phenotype. Mer- cells produced consistently higher levels of interstrand crosslinks than did Mer+ cells. In the present study we have measured the CNU-induced DNA interstrand crosslinking in IMR-90 normal human fibroblasts (Mer+), HT-29 human colon carcinoma cells (Mer+), and VA-13 SV-40 transformed human cells (Mer-) following pretreatment with MNNG. Cells were treated for 1 h with MNNG, then for an additional 1 h with CNU. Comparable levels of CNU-induced DNA interstrand crosslinking were observed in all cell lines. This crosslinking has been previously undetected in the IMR-90 and HT-29 cells. Cytotoxicity studies showed that MNNG pretreatment greatly enhanced the killing of IMR-90 and HT-29 cells by CNU, however, in VA-13 cells the increase in cell kill was smaller. These data suggest that in Mer+ cells a DNA repair system may remove chloroethyl monoadducts before the lethal DNA interstrand crosslinks can form. However, pretreatment of cells with MNNG may saturate this repair system rendering it inoperable.