File Name: cancer and cell cycle .zip
- Cell Cycle in Cancer
- Cell cycle checkpoint in cancer: a therapeutically targetable double-edged sword
- HHMI BioInteractive
- Cyclin A in cell cycle control and cancer
This interactive module explores the phases, checkpoints, and protein regulators of the cell cycle. The module also shows how mutations in genes that encode cell cycle regulators can lead to the development of cancer. Students can toggle between two different views of the cell cycle by pressing the text in the center of the graphic.
Cell Cycle in Cancer
Paclitaxel Taxol and carboplatin induce differential cell-cycle profiles in cell lines of head and neck squamous cell carcinoma. Content of DNA is represented on the x-axis; number of cells counted is represented on the y-axis. Data represent 3 independent experiments. Molecular analyses of cell-cycle regulatory proteins in head and neck squamous cell carcinoma HNSCC after paclitaxel Taxol and carboplatin treatment. Western blotting of p53 and p21 was performed.
Results represent 2 independent experiments. IP indicates immunoprecipitation. Effect of paclitaxel Taxol or carboplatin pretreatment on the cell-cycle kinetics of head and neck squamous cell carcinoma. Prepaclitaxel or precarboplatin indicates that the cells were pretreated with the drug for 8 hours before simultaneous treatment with both drugs. Molecular analyses of cell-cycle regulatory proteins in head and neck squamous cell carcinoma after paclitaxel Taxol and carboplatin pretreatment.
Pretreatment consisted of 8 hours' exposure to the first drug followed by the second drug as indicated, and protein was harvested. Effect of paclitaxel Taxol and carboplatin on the anchorage-independent growth of head and neck squamous cell carcinoma. Cells were treated in soft agar with paclitaxel alone, carboplatin alone, or simultaneously, or pretreated with carboplatin or paclitaxel for 8 hours in monolayer culture followed by soft-agar growth in the presence of both agents.
Colonies were grown for 14 days in soft agar before quantification. Results represent 8 independent determinations. Error bars represent SDs. Arch Otolaryngol Head Neck Surg. In contrast, carboplatin arrested cells before mitosis. Combination treatment with both agents, simultaneously or sequentially, was more effective at inhibiting cell growth than either single agent. Cellular outcome was the same regardless of which drug was used first.
The order of addition of these 2 drugs differentially affected cell-cycle position. Paclitaxel pretreatment arrested cells in mitosis, whereas carboplatin pretreatment or cotreatment resulted in premitotic arrest. These results provide molecular validation for the current clinical use of both drugs in combination and set the stage for analyses of patient tumor specimens.
Paclitaxel is a natural product from the bark of the western yew tree and one of a new class of agents known as taxanes. The effects of paclitaxel are correlated with tubulin polymerization and stabilization and subsequent arrest of the cells in mitosis. Normal eukaryotic cells progress through the cell cycle in a regulated manner owing to a cascade of biochemical events that coordinates the transition of cells from one phase to another.
During a normal cell cycle, the completion of mitosis is followed by the G 1 phase, in which a regulated series of events must take place before entry into the S phase. These events include elevations in D- and E-type cyclin levels, activation of cyclin-dependent kinases CDKs , phosphorylation of the retinoblastoma protein pRb , and subsequent activation of the E2F transcription factor family.
Cell-cycle transitions are regulated by checkpoint signaling pathways. These checkpoint pathways monitor cellular integrity and ensure the completion of one phase of the cell cycle before initiation of the next phase. When activated by various forms of cellular or genotoxic stress, checkpoint signaling can halt cell-cycle progression if abnormalities such as DNA damage, aneuploidy, or mitotic spindle anomalies exist.
During mitosis, the spindle checkpoint monitors spindle microtubule structure and chromosome alignment. In the current study, we explored how the chemotherapeutic agents paclitaxel and carboplatin modulate cell-cycle events in HNSCC cell lines. A panel of HNSCC cell lines were treated with various combinations of paclitaxel and carboplatin, and the effects on cell-cycle progression, checkpoint signaling pathways, and cell growth were examined.
Our primary objective was to gain insight into the mechanisms of drug action as they pertain to cell-cycle checkpoints in HNSCC. The ultimate goal is to use these preclinical molecular findings to devise improved clinical trials of paclitaxel and carboplatin and to provide clues to additional molecular mechanisms that can be targeted for the discovery of new drugs for the treatment of HNSCC. Dimethyl sulfoxide was used alone as a vehicle control.
Cells were treated with doxorubicin hydrochloride Adriamycin as indicated. The remaining cells were processed for protein analysis. Cellular proteins were prepared for Western blotting as previously described. Primary antibodies were detected using goat anti—mouse and rabbit anti—goat horseradish peroxidase—conjugated secondary antibodies Pierce, Rockford, Ill and subjected to enhanced chemiluminescence detection.
For each condition, protein extract was prepared. Values represent 2 independent experiments performed in quadruplicate. To determine the effects of paclitaxel and carboplatin on cell-cycle progression, we initially treated 2 HNSCC cell lines, UNC-7 and UM, with each drug and observed the effects for 72 hours by means of flow cytometry Figure 1.
Simultaneous treatment of the UM cells with both drugs also resulted in a similar cell-cycle profile to that observed with paclitaxel alone Figure 1. To gain insight into the molecular mechanisms behind the differential cell-cycle responses and outcomes observed after paclitaxel and carboplatin treatment, alone or in combination, we analyzed the drug-induced modulation of cell cycle and cell viability relative to the levels and activity of proteins involved in cell-cycle checkpoint signaling.
Cell-cycle and molecular analyses were performed at 6, 12, 24, and 36 hours after treatment. Carboplatin treatment stimulated an increase in the fraction of S-phase cells in all cell lines when used as a single agent. Thus, regardless of the differing genetic alterations present in the 4 tumor cell lines under examination, modulation of cell-cycle position was relatively similar after a given drug treatment.
These observations prompted an examination of the levels, phosphorylation status, and activity of select cell-cycle regulatory proteins. Cellular response to genotoxic agents and microtubule inhibitors has been linked to pmediated signaling.
Thus, we examined p53 protein levels and activity by means of Western blotting. We assessed p53 activity by looking for increased levels of the p53 downstream target gene product, p We observed a modest increase in p53 levels after 36 hours of carboplatin treatment, alone or in combination with paclitaxel, in the UNC-7 cells only Figure 2 A.
However, the increase in p53 level was not sufficient to induce expression of the downstream target p21, suggesting that the p53 signaling pathway is altered in this cell line. Levels of p53 were significantly elevated and not altered when compared with controls in the remaining lines, consistent with the presence of a mutant p53 protein Figure 2 B-D.
To further analyze the p53 status of the 4 cell lines, we compared, side by side, the basal p53 and p21 protein levels in all 4 HNSCC cell lines under control conditions Figure 2 E. In parallel, UNC-7 cells were treated with doxorubicin, an anticancer agent that induces stabilization of only wild-type p53 protein and elevation of p21 protein levels in cells containing functional, wild-type p Several studies indicate that the mitotic arrest mediated by microtubule inhibitors such as paclitaxel may result in cytotoxicity through alteration of normal mitotic signal transduction pathways, including prolonged CDC2 activity.
The cell-cycle arrest was accompanied by elevated levels of cyclin B1 protein and cyclin B1—immunoprecipitable kinase activity Figure 2 A-D. A previous study by Davis et al 35 has shown that the MPM-2 antibody recognizes phosphorylated protein epitopes found only in mitotic cells.
The length of the paclitaxel-mediated elevation in CDC2 activity varied among the different cell lines; however, a peak in activity preceded the accumulation of subdiploid cells that was observed at 24 hours after treatment.
Previously, sensitivity to anticancer agents has been shown to be influenced by alterations in posttranslational modifications of Bcl-2 family members, including the antiapoptotic protein Bcl Significant differences in the regulation of select cell-cycle signaling pathways accompanied the differential cell-cycle arrest induced by carboplatin in all the HNSCC cell lines Figure 2 A-D.
Between 24 and 36 hours, we saw a significant conversion of CDC2 from the hypophosphorylated, active form in the control cells to the hyperphosphorylated, inactive form of CDC2. Consistent with these changes in the phosphorylation status of CDC2 was an almost complete loss of cyclin B1—immunoprecipitable kinase activity and the absence of MPM-2 positivity on results of Western blotting.
Because simultaneous treatment of the HNSCC cells with paclitaxel and carboplatin led to an attenuation of mitotic arrest, we explored whether pretreatment of the cells with one drug before addition of the second would provide clues to the molecular changes that may be consistent with apoptosis.
To accomplish this, parallel experiments were designed in which HNSCC cell lines were subjected to one drug paclitaxel or carboplatin for 8 hours, followed by cotreatment with the second drug for 6, 12, 24, and 36 hours Figure 3. In the first set of experiments, cells were treated with paclitaxel for 8 hours, then treated with carboplatin.
The increase in S-phase fraction seen in the previous carboplatin treatments was reduced with this treatment regimen. The same lines were exposed to carboplatin for 8 hours before paclitaxel treatment. In the UM cells, there was a transient elevation in S-phase cell levels at 6 to 12 hours, followed by an arrest of cells with a 4N DNA content by 24 hours Figure 3. Modulation of p53 was not significantly different from that shown in the previous section.
In the UNC-7 cells, p53 levels increased with the carboplatin pretreatment schedule, but not with paclitaxel pretreatment Figure 4 A.
In the UM cells, a decrease in p21 protein was seen by 24 to 36 hours after pretreatment with paclitaxel or carboplatin. This latter change is likely the result of altered downstream p53 signaling. Again, as in the previous section, these differences did not significantly affect the cell-cycle kinetics. Coadministration resulted in the mitotic markers reverting from the mitotic form to the premitotic form between 12 and 24 hours.
In contrast, after paclitaxel pretreatment, the mitotic indices were still present at 24 hours, consistent with a prolonged mitotic arrest.
In the UMC cells, the patterns for simultaneous administration were similar to those for the paclitaxel pretreatment arm, which likely indicates cell-to-cell variability Figure 4 C. In the carboplatin pretreatment schedule, there was a conversion of CDC2 to the slower migrating, inactive form, a loss of cyclin B1 activity and MPM-2 positivity, and maintenance of Bcl-2 in the premitotic, dephosphorylated form Figure 4.
These data correlate with results of the flow cytometric analyses Figure 3 and suggest that these cells never entered mitosis. To extend the studies performed in monolayer and to further examine the chemosensitivity of the HNSCC cell lines, the effect of paclitaxel and carboplatin on anchorage-independent growth was assayed by means of growth in soft agar Figure 5. In 1 arm of the experiment, cells were treated in soft agar with paclitaxel or carboplatin alone or simultaneously.
Alternatively, cells were pretreated with paclitaxel or carboplatin for 8 hours in monolayer culture, followed by anchorage-independent growth in the presence of both agents. All 4 cell lines displayed the least sensitivity to carboplatin as a single agent. In the UNC and UMC cells, the combination of both agents resulted in a more significant reduction in colony number than use of either agent alone.
Overall, order of addition did not significantly affect outcome relative to simultaneous addition of both drugs in this assay. In an effort to increase effectiveness and decrease the toxicity of chemotherapeutic agents in the treatment of HNSCC, combination therapy has evolved. Until recently, these treatments have been reserved for inoperable or recurrent tumors.
By avoiding surgery, organ preservation and improved quality of life may be enjoyed by the patient. Several groups have demonstrated significant increases in response rates of head and neck tumors when treated with concomitant paclitaxel, carboplatin, and radiation therapies 14 - 16 , 41 ; however, improvement in survival has not yet been shown. The optimistic results of these phase 2 trials were the impetus for determining mechanism of action of these drugs in HNSCC cells.
We undertook the current study to delineate the response of 4 HNSCC cell lines to treatment with paclitaxel or carboplatin alone or in combination. Specifically, we examined the cell-cycle progression of these lines and determined the effect of these treatments on molecular mechanisms of cell-cycle control. However, regardless of the genetic alteration s that led to the loss of G 1 -phase checkpoint control, paclitaxel was more effective at inhibiting anchorage-independent cell growth compared with carboplatin, in all of the cell lines.
In contrast, carboplatin induced predominantly an S-phase arrest, except in the UM cells. Combination treatment, simultaneously or sequentially, was more effective than the use of either agent alone in the inhibition of HNSCC growth. Furthermore, when used in combination, the order of drug administration differentially affected cell-cycle position.
Cell cycle checkpoint in cancer: a therapeutically targetable double-edged sword
Pancreatic Cancer pp Cite as. All multicellular organisms arise from the division of a single cell. Thus, to generate a complex living organism, these cell divisions must be performed with extremely high fidelity and reproducibility during the development of the organism. Furthermore, in the mature, or adult, organism, tissue and organismal homeostasis must be maintained, and this requires the coordination of cell division with cell growth and cell death. These needs have led to the evolution of a cell replication process, known as the cell division cycle, that is highly conserved among all eukaryotes from simple single cellular organisms such as budding yeast to complex mammals such as humans. Pioneering studies by Lee Hartwell, performed in budding yeast, laid the groundwork for the identification and characterization of the key positive and negative regulators of this process.
Metrics details. Major currently used anticancer therapeutics either directly damage DNA or target and upset basic cell division mechanisms like DNA replication and chromosome segregation. These insults elicit activation of cell cycle checkpoints, safeguard mechanisms that cells implement to correctly complete cell cycle phases, repair damage or eventually commit suicide in case damage is unrepairable. Although cancer cells appear to be advantageously defective in some aspects of checkpoint physiology, recent acquisitions on the biochemical mechanisms of the various checkpoints are offering new therapeutic approaches against cancer. Indeed, chemical manipulation of these mechanisms is providing new therapeutic strategies and tools to increase the killing efficacy of major cancer therapeutics as well as to directly promote cancer cell death. In this review we summarize developing concepts on how targeting cell cycle checkpoints may provide substantial improvement to cancer therapy.
The cell cycle , or cell-division cycle , is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA DNA replication and some of its organelles , and subsequently the partitioning of its cytoplasm and other components into two daughter cells in a process called cell division. In cells with nuclei eukaryotes , i. During interphase, the cell grows, accumulating nutrients needed for mitosis, and replicates its DNA and some of its organelles. During the mitotic phase, the replicated chromosomes, organelles, and cytoplasm separate into two new daughter cells.
Cyclin A in cell cycle control and cancer
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The cell cycle, the process by which cells progress and divide, lies at the heart of cancer. In normal cells, the cell cycle is controlled by a complex series of signaling pathways by which a cell grows, replicates its DNA and divides. This process also includes mechanisms to ensure errors are corrected, and if not, the cells commit suicide apoptosis. In cancer, as a result of genetic mutations, this regulatory process malfunctions, resulting in uncontrolled cell proliferation. Cyclacel Pharmaceuticals' drug discovery and development programs build on scientific advances in understanding these molecular mechanisms. Through our expertise, we are developing cell cycle-based, mechanism-targeted cancer therapies that emulate the body's natural process in order to stop the growth of cancer cells. This approach can limit the damage to normal cells and the accompanying side effects caused by conventional chemotherapeutic agents.
Cyclin A is particularly interesting among the cyclin family because it can activate two different cyclin-dependent kinases CDKs and functions in both S phase and mitosis. An embryonic form of cyclin A that is only essential for spermatogenesis is also present in some organisms. In mitosis, the precise role of cyclin A is still obscure, but it may contribute to the control of cyclin B stability. Cyclin A starts to accumulate during S phase and is abruptly destroyed before metaphase. The synthesis of cyclin A is mainly controlled at the transcription level, involving E2F and other transcription factors. Consistent with its role as a key cell cycle regulator, expression of cyclin A is found to be elevated in a variety of tumors.
Article · Figures & SI · Info & Metrics · PDF. Loading. Recent insights in the fields of cell cycle regulation and cancer would each alone have provided prime.
This is an open access article distributed under the terms of Creative Commons Attribution License. Decades of research in the fields of biochemistry, cell biology, molecular genetics, virology, genetic engineering, functional genomics and cancer gene therapy, have converged in identifying the executive components of a commanding regulatory axis of mammalian cell cycle control: Providing new mechanistic insights, biochemical pathways, unifying concepts and checkpoint control elements with profound implications in the prevention, diagnosis and treatment of cancer Fig. The challenge remains, however, to successfully integrate these celebrated epochs of gene discovery and biochemical pathway characterization into a practical understanding of cell cycle control befitting the actual praxis and applied pharmacology of contemporary clinical oncologists. This focused review, prepared by contributing scientists and clinical practitioners in the field of genetic medicine, is intended to present the current state-of-the-art in applied cell cycle checkpoint control as it relates to cancer management. From a clinical perspective, the molecular mechanisms of chemical co-carcinogenesis first came to light in the s with the pioneering studies of croton oil i. The subsequent discovery that protein kinase C PKC , which plays a major role in signal transduction and cell proliferation, is the cellular receptor for the tumor-promoting phorbol esters 3 ushered in a wave of pharmaceutical interest in selective PKC inhibitors, only to be thwarted by the general multifunctionality of PKC, the multiplicity of PKC isoenzymes, the limited specificity of PKC modulators and the remaining unanswered questions and intricacies of PKC function, which stifled the promise of targeting PKCs for cancer therapeutics 4. Basic research in yeast genetics characterized a number of cell division cycle Cdc mutants, thereby identifying important genes, notably Cdc28 in baker's yeast S.
Cancer is characterized by uncontrolled proliferation resulting from aberrant activity of various cell cycle proteins; therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrated that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. In contrast, many cancers are uniquely dependent on these proteins and are hence selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. Here, we review the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as future therapeutic potential of various inhibitors. We focus only on proteins that directly regulate cell cycle progression.
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