Simultaneous measurement of passage through the restriction point and MCM loading in single cells.

Passage through the Retinoblastoma protein (RB1)-dependent restriction point and the loading of minichromosome maintenance proteins (MCMs) are two crucial events in G1-phase that help maintain genome integrity. Deregulation of these processes can cause uncontrolled proliferation and cancer development. Both events have been extensively characterized individually, but their relative timing and inter-dependence remain less clear. Here, we describe a novel method to simultaneously measure MCM loading and passage through the restriction point. We exploit that the RB1 protein is anchored in G1-phase but is released when hyper-phosphorylated at the restriction point. After extracting cells with salt and detergent before fixation we can simultaneously measure, by flow cytometry, the loading of MCMs onto chromatin and RB1 binding to determine the order of the two events in individual cells. We have used this method to examine the relative timing of the two events in human cells. Whereas in BJ fibroblasts released from G0-phase MCM loading started mainly after the restriction point, in a significant fraction of exponentially growing BJ and U2OS osteosarcoma cells MCMs were loaded in G1-phase with RB1 anchored, demonstrating that MCM loading can also start before the restriction point. These results were supported by measurements in synchronized U2OS cells.

p53-dependent G(1) arrest in 1st or 2nd cell cycle may protect human cancer cells from cell death after treatment with ionizing radiation and Chk1 inhibitors.

OBJECTIVES: This study was performed to explore the strategy of combining Chk1 inhibitors with ionizing radiation (IR) to selectively target p53-deficient cancer cells. MATERIALS AND METHODS: Survival and cell cycle progression were measured in response to IR and the Chk1 inhibitors, UCN-01 and CEP-3891, in colon carcinoma HCT116 p53+/+ and p53-/- cells, and in osteosarcoma U2OS-VP16 cells with conditional expression of dominant-negative p53 (p53DD). RESULTS: Clonogenic survival was selectively reduced in HCT116 p53-/- compared to p53+/+ cells after treatment with UCN-01 and IR, and HCT116 p53+/+ cells also displayed strong p53-dependent G(1) arrest in the 1st cell cycle after IR. In contrast, clonogenic survival was affected similarly in U2OS-VP16 cells with and without expression of p53DD. However, death of U2OS-VP16 cells was p53 dependent as assessed by cell viability assay at 72 h, and this was associated with p53-dependent G(1) arrest in the 2nd cell cycle after treatment. Notably, HCT116 cells were overall more resistant than U2OS cells to cytotoxic effects of Chk1 inhibitors. CONCLUSION: Our results suggest that p53-dependent G(1) arrest in both 1st and 2nd cell cycles may protect human cancer cells from cell death after treatment with IR and Chk1 inhibitors. However, a challenge for future clinical use will be that different cancers display different intrinsic sensitivity to such inhibitors.

Checkpoint adaptation in human cells.

Checkpoint adaptation was originally described in Saccharomyces cerevisiae as the ability to divide following a sustained checkpoint arrest in the presence of unrepairable DNA breaks. A process of checkpoint adaptation was also reported in Xenopus in response to the replication inhibitor aphidicolin. Recently, we showed that checkpoint adaptation also occurs in human cells. Although cells undergoing checkpoint adaptation will frequently die in subsequent cell cycles owing to excessive DNA damage, some of the cells may be able to survive and proliferate with damaged DNA. Thus, checkpoint adaptation in human cells may potentially promote genomic instability and lead to cancer. Here, I discuss the current evidence for checkpoint adaptation in human cells and possible mechanisms and implications of this phenomenon.

The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.

When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the IR-induced S-phase checkpoint cause 'radioresistant DNA synthesis', a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene. The Cdc25A phosphatase activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. Here we report a functional link between ATM, the checkpoint signalling kinase Chk2/Cds1 (Chk2) and Cdc25A, and implicate this mechanism in controlling the S-phase checkpoint. We show that IR-induced destruction of Cdc25A requires both ATM and the Chk2-mediated phosphorylation of Cdc25A on serine 123. An IR-induced loss of Cdc25A protein prevents dephosphorylation of Cdk2 and leads to a transient blockade of DNA replication. We also show that tumour-associated Chk2 alleles cannot bind or phosphorylate Cdc25A, and that cells expressing these Chk2 alleles, elevated Cdc25A or a Cdk2 mutant unable to undergo inhibitory phosphorylation (Cdk2AF) fail to inhibit DNA synthesis when irradiated. These results support Chk2 as a candidate tumour suppressor, and identify the ATM-Chk2-Cdc25A-Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis.

Molecular events in radiation transformation.

Studies of human tumor cell lines have revealed alterations in the regulation of a number of cell cycle-related genes, associated in some cases with a TP53-independent loss of the radiation-induced G(1)-phase arrest. It is not clear, however, whether these are early or late events in tumor development, or they arise in tumor cell lines during growth in culture. Since the oncogenic transformation of an individual cell is thought to be an early event in tumor development, we have used a model system of normal and radiation-transformed C3H 10T(1/2) mouse fibroblast cell clones to address this issue. Transformed clones derived from type III foci were compared with clones derived from parental, wild-type cells. Approximately 25% of transformed clones showed Trp53 mutations in exon 5; however, preliminary results based on in situ immunofluorescence studies with an antibody recognizing mutant Trp53 indicate that the appearance of such mutations in transformed clones occurs late in the process of transformation and is unlikely to represent an initiating event. The remaining transformed clones and all clones derived from parental cells expressed wild-type Trp53. Radiation-induced G(1)-phase arrest was either absent or significantly reduced in all of the transformed clones, independent of Trp53 status. Constitutive expression of Cdkn1a protein was significantly increased in most of the transformed clones. Also, the majority of transformed clones showed elevated levels of cyclin D1, and two clones overexpressed cyclin E. These results indicate that loss of G(1)-phase checkpoint control, independent of Trp53 status, and altered expression of cell cycle regulatory proteins may represent early events in the process of radiation-induced carcinogenesis that are associated with the malignant transformation of individual cells.

Rapid destruction of human Cdc25A in response to DNA damage.

To protect genome integrity and ensure survival, eukaryotic cells exposed to genotoxic stress cease proliferating to provide time for DNA repair. Human cells responded to ultraviolet light or ionizing radiation by rapid, ubiquitin- and proteasome-dependent protein degradation of Cdc25A, a phosphatase that is required for progression from G1 to S phase of the cell cycle. This response involved activated Chk1 protein kinase but not the p53 pathway, and the persisting inhibitory tyrosine phosphorylation of Cdk2 blocked entry into S phase and DNA replication. Overexpression of Cdc25A bypassed this mechanism, leading to enhanced DNA damage and decreased cell survival. These results identify specific degradation of Cdc25A as part of the DNA damage checkpoint mechanism and suggest how Cdc25A overexpression in human cancers might contribute to tumorigenesis.

c-Abl is required for development and optimal cell proliferation in the context of p53 deficiency.

The c-Abl tyrosine kinase and the p53 tumor suppressor protein interact functionally and biochemically in cellular genotoxic stress response pathways and are implicated as downstream mediators of ATM (ataxia-telangiectasia mutated). This fact led us to study genetic interactions in vivo between c-Abl and p53 by examining the phenotype of mice and cells deficient in both proteins. c-Abl-null mice show high neonatal mortality and decreased B lymphocytes, whereas p53-null mice are prone to tumor development. Surprisingly, mice doubly deficient in both c-Abl and p53 are not viable, suggesting that c-Abl and p53 together contribute to an essential function required for normal development. Fibroblasts lacking both c-Abl and p53 were similar to fibroblasts deficient in p53 alone, showing loss of the G(1)/S cell-cycle checkpoint and similar clonogenic survival after ionizing radiation. Fibroblasts deficient in both c-Abl and p53 show reduced growth in culture, as manifested by reduction in the rate of proliferation, saturation density, and colony formation, compared with fibroblasts lacking p53 alone. This defect could be restored by reconstitution of c-Abl expression. Taken together, these results indicate that the ATM phenotype cannot be explained solely by loss of c-Abl and p53 and that c-Abl contributes to enhanced proliferation of p53-deficient cells. Inhibition of c-Abl function may be a therapeutic strategy to target p53-deficient cells selectively.

Radiation-induced apoptosis and cell cycle progression in Jurkat T cells.

The purpose of this study was to explore the connection between radiation-induced apoptosis and progression of cells through the phases of the cell cycle. Cells of the human T-cell line Jurkat were separated by centrifugal elutriation into populations enriched in G(1)-, S- and G(2)/M-phase cells before irradiation. After a dose of 20 Gy, the onset of massive apoptosis occurred at about 6 h in all populations regardless of the phase of the cell cycle in which they were irradiated. In contrast, after 2 Gy, cells died at various times after a pronounced G(2)/M-phase arrest. These results indicate that radiation-induced apoptosis can occur independently of cell cycle arrest and that the time for onset of apoptosis may be dependent on the radiation dose.

Loss of normal G1 checkpoint control is an early step in carcinogenesis, independent of p53 status.

Recent studies have described a diminished radiation-induced G1 arrest in some wild-type (wt) p53 human tumor cell lines compared to normal human fibroblasts. However, the significance of this finding was unclear, particularly because tumor cell lines may have accumulated additional genetic changes after long periods in culture. Because malignant transformation of individual cells is thought to be an early step in carcinogenesis, we have used a model system of normal and transformed mouse fibroblast 10T1/2 cell clones to examine whether loss of G1 checkpoint control may be an early event in tumor development and to study the relationships between G1 arrest, radiosensitivity, and genetic alterations. Twelve transformed clones were established from type III foci induced by irradiation of normal 10T1/2 cells and were compared with six clones derived from wt 10T1/2 cells. Three of the transformed clones expressed mutant p53; two of these had the same point mutation at codon 132 (exon 5), and one had a point mutation at codon 135. The remaining transformed and normal clones had wt p53 status. The radiosensitivity of transformed clones, as measured by a clonogenic assay, was similar to that of normal clones; the three clones with mutant p53 did not differ from the others. There was no relationship between G1 arrest and radiosensitivity. Normal 10T1/2 cell clones showed a transient G1 arrest lasting approximately 9 h after 6 Gy of irradiation. This G1 arrest was either absent or markedly reduced in all of the transformed clones, regardless of p53 status. These results suggest that diminished G1 checkpoint control is an early event in the process of carcinogenesis that is associated with the malignant transformation of individual cells and is independent of p53 status.

Sensitization of renal carcinoma to radiation using alpha interferon (IFNA) gene transfection.

The rationale for this study was that local delivery of interferon-alpha (IFN-alpha) by gene transfection may be of value during radiotherapy. To investigate the feasibility of this approach, cells of the human renal carcinoma cell line R11 were transfected with the IFNA gene and evaluated for radiation responses in vitro by clonogenic assays. R11 cells expressing IFN-alpha after gene transfection were more sensitive to radiation than R11 control cells (SF2 = 0.33 and 0.51, respectively). In addition to increasing radiosensitivity, IFNA gene transfection slowed cellular growth and reduced the plating efficiency in clonogenic assays. The addition of exogenous rhIFN-alpha to cells at different times relative to irradiation showed that its presence during the postirradiation period was critical for radiosensitization, but repair of sublethal damage did not seem to be affected. No apoptosis of R11 cells was found 1-5 days after exposure to 2-25 Gy with or without IFN-alpha. Extensive formation of multinuclear giant cells was present beginning 2 days after irradiation; however, IFN-alpha did not cause any major alterations in the yield of radiation-induced giant cells. These studies suggest that gene transfection might be an effective means of delivering IFN-alpha for clinical use in radiotherapy of cancer.

Effects of IL-3 gene expression on tumor response to irradiation in vitro and in vivo.

Expression of a murine interleukin 3 gene in murine fibrosarcoma cells (FSA-JmIL-3) did not alter their survival after in vitro irradiation. However, FSA-JmIL-3 tumors established in vivo were much more sensitive to irradiation than was the parental tumor. Following 25 Gy of irradiation, parental fibrosarcoma tumors regrew after a growth delay of 10 days, but FSA-JmIL-3 tumors continued to regress. Examination of the cellular composition of tumors following irradiation revealed that, instead of tumor cell repopulation, the FSA-JmIL-3 tumors became heavily infiltrated with lymphocytes, indicating that the effect of irradiation was to allow the IL-3-elicited cellular immune response to infiltrate the tumors and mediate rejection. This study indicates that combining gene immunotherapy approaches with radiotherapy might increase the effectiveness of both, and it seems logical to pursue such treatment options.

Apoptosis and delayed expression of c-jun and c-fos after gamma irradiation of Jurkat T cells.

The purpose of this study was to determine the role of radiation-induced expression of c-jun and c-fos in radiation-induced apoptosis of cells of the Jurkat T-cell line. Doses of 10-20 Gy caused a massive number of cells to undergo apoptosis within the first 24 h. This was accompanied by extensive increases in c-jun mRNA levels and moderate increases in c-fos levels, both occurring at the time of onset of internucleosomal DNA fragmentation. Increased c-jun and c-fos expression was maximum at 8 h after irradiation with a 10-fold increase in c-jun and a 2-fold increase in c-fos mRNA levels. In comparison, stimulation of the Jurkat cells with PMA resulted in rapid induction of c-jun and c-fos within 1 h. The late induction of c-jun and c-fos was not preceded by induction of tumor necrosis factor-alpha (TNF-alpha) or the bifunctional repair endonuclease and nuclear redox factor Ref-1; rather a slow decrease in Ref-1 mRNA levels was found over the first 24 h. Our results showed that radiation-induced c-jun and c-fos expression is a late response in Jurkat cells, and is most likely a secondary effect not necessary for radiation-induced apoptosis. Furthermore, apoptosis was induced by the RNA synthesis inhibitor actinomycin D, which does not induce c-jun or c-fos expression. This demonstrates that massive late induction of c-jun and c-fos is not a general requirement for apoptosis in Jurkat cells.

The effects of cytokine gene transfer into tumors on host cell infiltration and regression.

New strategies are becoming available that promise to revolutionize cancer immunotherapy. Although the task of generating what is in essence a pathogenic autoimmune anti-tumor response in the face of local and systemic immune suppression is likely to remain a formidable one, advances in molecular strategies for enhancing tumor immunity have been made that show considerable promise, in particular those based on gene transfer technology. For example, introduction of certain cytokine genes into murine tumor cells have been shown to enhance tumor immunogenicity and induce regression. Caution is needed in properly interpreting the relevance of observations derived from murine models for human cancer, but clinical trials are underway that will test the utility of cytokine gene therapy for cancer and that will generate data that will be useful for the design of future strategies. Because of the magnitude of the problem of inducing tumor regression, it is argued that, even if genetically engineering can be used to successfully enhance anti-tumor immunity, combination of such strategies with other existing conventional anti-cancer therapies, that increase the effectiveness of both, may be necessary to reliably achieve cure.