William K. Kaufmann

p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest

gamma-Irradiation of human diploid fibroblasts in the G1 interval caused arrest of the cell cycle prior to S phase. This cell cycle block was correlated with a lack of activation of both cyclin E-Cyclin-dependent kinase 2 (Cdk2) and cyclin A-Cdk2 kinases and depended on wild-type p53. Although the accumulation of cyclin A was strongly inhibited in gamma-irradiated cells, cyclin E accumulated and bound Cdk2 at normal levels but remained in an inactive state. We found that both whole-cell lysates and inactive cyclin E-Cdk2 complexes prepared from irradiated cells contained an activity capable of inactivating cyclin E-Cdk2 complexes. The protein responsible for this activity was shown to be p21CIP1/WAF1, recently described as a p53-inducible Cdk inhibitor. Our data suggest a model in which ionizing radiation confers G1 arrest via the p53-mediated induction of a Cdk inhibitor protein.

Oxidative stress and cell cycle checkpoint function.

Oxidative stress and the damage that results from it have been implicated in a wide number of disease processes including atherosclerosis, autoimmune disorders, neuronal degeneration, and cancer. Reactive oxygen species (ROS) are ubiquitous and occur naturally in all aerobic species, coming from both exogenous and endogenous sources. ROS are quite reactive and readily damage biological molecules, including DNA. While the damaging effects of ROS on DNA have been intensively studied, the effects of oxidative damage on cell cycle checkpoint function have not. Here will we review several biologically important ROS and their sources, the cell cycle, checkpoints, and current knowledge about the effects of ROS on initiating checkpoint responses.Oxidative stress and the damage that results from it have been implicated in a wide number of disease processes including atherosclerosis, autoimmune disorders, neuronal degeneration, and cancer. Reactive oxygen species (ROS) are ubiquitous and occur naturally in all aerobic species, coming from both exogenous and endogenous sources. ROS are quite reactive and readily damage biological molecules, including DNA. While the damaging effects of ROS on DNA have been intensively studied, the effects of oxidative damage on cell cycle checkpoint function have not. Here will we review several biologically important ROS and their sources, the cell cycle, checkpoints, and current knowledge about the effects of ROS on initiating checkpoint responses.

DNA damage and cell cycle checkpoints.

DNA is prone to numerous forms of damage that can injure cells and impair fitness. Cells have evolved an array of mechanisms to repair these injuries. Proliferating cells are especially vulnerable to DNA damage due to the added demands of cellular growth and division. Cell cycle checkpoints represent integral components of DNA repair that coordinate cooperation between the machinery of the cell cycle and several biochemical pathways that respond to damage and restore DNA structure. By delaying progression through the cell cycle, checkpoints provide more time for repair before the critical phases of DNA replication, when the genome is replicated, and of mitosis, when the genome is segregated. Loss or attenuation of checkpoint function may increase spontaneous and induced gene mutations and chromosomal aberrations by reducing the efficiency of DNA repair. Defects in checkpoint control have been seen in certain hereditary cancer syndromes and at early stages of cell transformation. Mutations in checkpoint control genes therefore may contribute to the genetic instability that appears to drive neoplastic evolution.—Kaufmann, W. K., Paules, R. S. DNA damage and cell cycle checkpoints. FASEB J. 10, 238‐247 (1996)

Coupling of Human Circadian and Cell Cycles by the Timeless Protein

ABSTRACT The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. We have found that the human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein.

An ATR- and Chk1-Dependent S Checkpoint Inhibits Replicon Initiation following UVC-Induced DNA Damage

ABSTRACT Inhibition of replicon initiation is a stereotypic DNA damage response mediated through S checkpoint mechanisms not yet fully understood. Studies were undertaken to elucidate the function of checkpoint proteins in the inhibition of replicon initiation following irradiation with 254 nm UV light (UVC) of diploid human fibroblasts immortalized by the ectopic expression of telomerase. Velocity sedimentation analysis of nascent DNA molecules revealed a 50% inhibition of replicon initiation when normal human fibroblasts were treated with a low dose of UVC (1 J/m2). Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and AT-like disorder fibroblasts, which lack an S checkpoint response when exposed to ionizing radiation, responded normally when exposed to UVC and inhibited replicon initiation. Pretreatment of normal and AT fibroblasts with caffeine or UCN-01, inhibitors of ATR (AT mutated and Rad3 related) and Chk1, respectively, abolished the S checkpoint response to UVC. Moreover, overexpression of kinase-inactive ATR in U2OS cells severely attenuated UVC-induced Chk1 phosphorylation and reversed the UVC-induced inhibition of replicon initiation, as did overexpression of kinase-inactive Chk1. Taken together, these data suggest that the UVC-induced S checkpoint response of inhibition of replicon initiation is mediated by ATR signaling through Chk-1 and is independent of ATM, Nbs1, and Mre11.

The Human Tim/Tipin Complex Coordinates an Intra-S Checkpoint Response to UV That Slows Replication Fork Displacement

ABSTRACT UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m2 UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

The human decatenation checkpoint

Chromatid catenation is actively monitored in human cells, with progression from G2 to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATRki). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G2 checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATRki produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.

Cell-Type-Specific Responses to Chemotherapeutics in Breast Cancer

Recent microarray studies have identified distinct subtypes of breast tumors that arise from different cell types and that show statistically significant differences in patient outcome. To gain insight into these differences, we identified in vitro and in vivo changes in gene expression induced by chemotherapeutics. We treated two cell lines derived from basal epithelium (immortalized human mammary epithelial cells) and two lines derived from luminal epithelium (MCF-7 and ZR-75–1) with chemotherapeutics used in the treatment of breast cancer and assayed for changes in gene expression using DNA microarrays. Treatment doses for doxorubicin and 5-fluorouracil were selected to cause comparable cytotoxicity across all four cell lines. The dominant expression response in each of the cell lines was a general stress response; however, distinct expression patterns were observed. Both cell types induced DNA damage-response genes such as p21waf1, but the response in the luminal cells showed higher fold changes and included more p53-regulated genes. Luminal cell lines repressed a large number of cell cycle-regulated genes and other genes involved in cellular proliferation, whereas the basal cell lines did not. Instead, the basal cell lines repressed genes that were involved in differentiation. These in vitro responses were compared with expression responses in breast tumors sampled before and after treatment with doxorubicin or 5-fluorouracil/mitomycin C. The in vivo data corroborated the cell-type-specific responses to chemotherapeutics observed in vitro, including the induction of p21waf1. Similarities between in vivo and in vitro responses help to identify important response mechanisms to chemotherapeutics.

Control of skin cancer by the circadian rhythm

Skin cancer is the most common form of cancer in the United States. The main cause of this cancer is DNA damage induced by the UV component of sunlight. In humans and mice, UV damage is removed by the nucleotide excision repair system. Here, we report that a rate-limiting subunit of excision repair, the xeroderma pigmentosum group A (XPA) protein, and the excision repair rate exhibit daily rhythmicity in mouse skin, with a minimum in the morning and a maximum in the afternoon/evening. In parallel with the rhythmicity of repair rate, we find that mice exposed to UV radiation (UVR) at 4:00 AM display a decreased latency and about a fivefold increased multiplicity of skin cancer (invasive squamous cell carcinoma) than mice exposed to UVR at 4:00 PM. We conclude that time of day of exposure to UVR is a contributing factor to its carcinogenicity in mice, and possibly in humans.

Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032

PURPOSE To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells. MATERIALS AND METHODS A large collection of melanoma cell lines (n=37) were treated with 0-8Gy IR and clonogenic survival assays used to generate survival curves to rank relative radiosensitivities among the cell lines. The ability of a B-RAF inhibitor, PLX-4032, to radiosensitize highly radioresistant B-Raf+ cells was also assessed by clonogenic cell survival and spheroid invasion assays and the effects of treatment on the cell cycle assessed by FACS. RESULTS Melanoma cell lines displayed a very large, heterogeneous range of SF2 values (1.002-0.053) with a mean of 0.51. Cell lines with surviving fractions of 0.29 or less at SF2 and SF4 were observed at a high frequency of 18.9% and 70.2%, respectively. Treatment of B-Raf+ cells with the B-RAF inhibitor PLX-4032 in combination with radiation provided enhanced inhibition of both colony formation and invasion, and radiosensitized cells through an increase in G(1) arrest. CONCLUSIONS Our data suggest that melanomas are not uniformly radioresistant with a significant subset displaying inherent radiosensitivity. Pharmacologic inhibition of B-RAF with PLX-4032 effectively radiosensitized B-Raf+ melanoma cells suggesting that this combination approach could provide improved radiotherapeutic response in B-Raf+ melanoma patients.