Cell cycle regulators
- 2. FIRSTUP CONSULTANTS INDEX 1. INTRODUCTION 2. ACTIVATORS AND INHIBITORS 3. ALTERATIONS IN CELL CYCLE CONTROL PROTEINS IN CANCER CELLS 4. INSENSITIVITY TO GROWTH INHIBITORY SIGNALS 5. RB GENE: Governor Of The Cell Cycle 6. TP53 GENE: Guardian Of The Genome 7. TRANSFORMING GROWTH FACTOR-B PATHWAY 8. APC-B-CATENIN PATHWAYS 9. REFERENCES
- 3. INTRODUCTION
- 4. FIRSTUP CONSULTANTS Cell proliferation is fundamental to: 1. Development. 2. Maintenance of steady-state tissue homeostasis. 3. Replacement of dead or damaged cells. The key elements of cellular proliferation to daughter cells through mitosis and cytokinesis are: 1. Accurate DNA replication. 2. Coordinated synthesis of all other cellular constituents. 3. Equal apportionment of DNA and other cellular organelles.
- 6. FIRSTUP CONSULTANTS The sequence of events that results in cell division is called the cell cycle. It consists of: 1. G1 (Presynthetic growth) Phase 2. S (DNA synthesis) Phase 3. G2 (Premitotic growth) Phase 4. M (Mitotic) Phase Quiescent cells that are not actively cycling are said to be in the G0 state. Cells can enter G1 either from the G0 quiescent cell pool, or after completing a round of mitosis, as for continuously replicating cells. Non-fidelity of DNA replication, or cofactor deficiency result in arrest at the various transition points.
- 9. FIRSTUP CONSULTANTS Cell cycle progression is driven by: 1. Proteins called cyclins named for the cyclic nature of their production and degradation. 2. Cyclin-associated enzymes called cyclin dependent kinases (CDKs). CDKs acquire the ability to phosphorylate protein substrates by forming complexes with the relevant cyclins. Transiently increased synthesis of a particular cyclin leads to increased kinase activity of the appropriate CDK binding partner. As the CDK completes its round of phosphorylation, the associated cyclin is degraded and the CDK activity abates. Thus, as cyclin levels rise and fall, the activity of associated CDKs likewise wax and wane.
- 11. FIRSTUP CONSULTANTS Embedded in the cell cycle are surveillance mechanisms primed to sense DNA or chromosomal damage. These quality control checkpoints ensure that cells with genetic imperfections do not complete replication: 1. The G1-S checkpoint monitors the integrity of DNA before irreversibly committing cellular resources to DNA replication. 2. The G2-M restriction point ensures that there has been accurate genetic replication before the cell actually divides. When cells do detect DNA irregularities, checkpoint activation delays cell cycle progression and triggers DNA repair mechanisms. If the genetic derangement is too severe to be repaired, the cells will undergo apoptosis; alternatively, they may enter a non replicative state called senescence.
- 13. FIRSTUP CONSULTANTS Enforcing the cell cycle checkpoints is the job of CDK inhibitors(CDKIs). There are several different CDKIs: 1. The Cip/Kip (CDK interacting protein/Kinase inhibitory protein) family broadly inhibits multiple CDKs and includes: a) p21 (CDKN1A) b) p27 (CDKN1B) c) p57 (CDKN1C) 2. The INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) family has selective effects on cyclin CDK4 and cyclin CDK6 and includes: a) p15 (CDKN2B) b) p16 (CDKN2A) c) p18 (CDKN2C) d) p19 (CDKN2D)
- 15. FIRSTUP CONSULTANTS While cyclins arouse the CDKs, CDK inhibitors (CDKIs), of which there are many, silence the CDKs and exert negative control over the cell cycle. Defective CDKI checkpoint proteins allow cells with damaged DNA to divide, resulting in mutated daughter cells with the potential of developing into malignant tumors.
- 17. FIRSTUP CONSULTANTS The major cancer-associated mutations that affect the G1/S checkpoint can be broadly grouped into two classes: 1. Gain-of-function mutations involving CDK4 and D cyclin: a) Mishaps increasing the expression of cyclin D or CDK4 are common events in neoplastic transformation. b) The cyclin D genes are overexpressed in many cancers. 2. Loss-of-function mutations involving CDKIs: a) CDKIs that inhibit cyclin D/CDK complexes are frequently mutated or otherwise silenced in many human malignancies.
- 19. FIRSTUP CONSULTANTS In principle, anti-growth signals can prevent cell proliferation by several complementary mechanisms. The signal may cause dividing cells to enter G0, where they remain until external cues prod their re-entry into the proliferative pool. Alternatively, the cells may enter a postmitotic, differentiated pool and lose replicative potential. Non-replicative senescence, is another mechanism of escape from sustained cell growth. And, as a last-ditch effort, the cells may be programmed for death by apoptosis. Therefore, tumor suppressor genes have all these “tricks” in their toolbox designed to halt wayward cells from becoming malignant.
- 21. FIRSTUP CONSULTANTS RB, a key negative regulator of the cell cycle, is directly or indirectly inactivated in most human cancers. The retinoblastoma gene (RB) was the first tumor suppressor gene to be discovered. The discovery of tumor suppressor genes was accomplished by the study of a rare disease—in this case, retinoblastoma, an uncommon childhood tumor. The tumor is being transmitted as an autosomal dominant trait. It is of two types: 1. 60% sporadic 2. 40% familial
- 23. FIRSTUP CONSULTANTS To account for the sporadic and familial occurrence of an identical tumor, Knudson, in 1974, proposed his now famous two-hit hypothesis, which in molecular terms can be stated as follows: 1. Two mutations (hits) are required to produce retinoblastoma. 2. These involve the RB gene, which has been mapped to chromosomal locus 13q14. 3. Both of the normal alleles of the RB locus must be inactivated for the development of retinoblastoma.
- 24. Alfred George Knudson, Jr. (August 9, 1922 – July 10, 2016)
- 26. FIRSTUP CONSULTANTS In Sporadic Cases: 1. Both normal RB alleles are lost by somatic mutation in one of the retinoblasts. 2. In the end a retinal cell that has lost both of the normal copies of the RB gene becomes cancerous. In Familial Cases: 1. Children inherit one defective copy of the RB gene in the germ line; the other copy is normal. 2. Retinoblastoma develops when the normal RB gene is lost in retinoblasts as a result of somatic mutation. 3. Because in retinoblastoma families a single germ line mutation is sufficient to transmit disease risk, the trait has an autosomal dominant inheritance pattern.
- 28. FIRSTUP CONSULTANTS Although the loss of normal RB genes initially was discovered in retinoblastomas, it is now evident that biallelic loss of this gene is a fairly common feature of several tumors like: 1. Breast cancer 2. Small cell cancer of the lung 3. Bladder cancer Patients with familial retinoblastoma also are at greatly increased risk for developing: 1. Osteosarcomas 2. Some soft-tissue sarcomas.
- 29. BREAST CANCER SMALL CELL CANCER OF THE LUNG BLADDER CANCER OSTEOSARCOMA SOFT TISSUE SARCOMA
- 30. FIRSTUP CONSULTANTS The function of the RB protein is to regulate the G1/S checkpoint, the portal through which cells must pass before DNA replication commences. In the G1 phase, diverse signals are integrated to determine whether the cell should progress through the cell cycle, or exit the cell cycle and differentiate. RB, is a DNA-binding protein that serves as a point of integration for these diverse signals, which ultimately act by altering the phosphorylation state of RB. Specifically, signals that promote cell cycle progression lead to the phosphorylation and inactivation of RB, while those that block cell cycle progression act by maintaining RB in an active hypophosphorylated state.
- 31. FIRSTUP CONSULTANTS Mechanism: 1. The initiation of DNA replication requires the activity of cyclin E/CDK2 complexes, and expression of cyclin E is dependent on the E2F(Elongation Factor 2) family of transcription factors. 2. Early in G1, RB is in its hypophosphorylated active form, and it binds to and inhibits the E2F family of transcription factors, preventing transcription of cyclin E. 3. Hypophosphorylated RB blocks E2F-mediated transcription in 2 ways: a) It sequesters E2F, preventing it from interacting with other transcriptional activators. b) RB recruits chromatin remodeling proteins to make DNA insensitive to transcription factors, such as histone deacetylases and histone methyltransferases, which bind to the promoters of E2F-responsive genes such as cyclin E.
- 33. FIRSTUP CONSULTANTS This situation is changed on mitogenic signaling: 1. Growth factor signaling leads to cyclin D expression and activation of cyclin D–CDK4/6 complexes. 2. These complexes phosphorylate RB, inactivating the protein and releasing E2F to induce target genes such as cyclin E. 3. Cyclin E/CDK complexes then stimulate DNA replication and progression through the cell cycle. 4. When the cells enter S phase, they are committed to divide without additional growth factor stimulation. 5. During the ensuing M phase, the phosphate groups are removed from RB by cellular phosphatases, regenerating the hypophosphorylated form of RB.
- 35. FIRSTUP CONSULTANTS Q: Why RB is not mutated in every cancer? Mutations in other genes that control RB phosphorylation can mimic the effect of RB loss and are commonly found in many cancers that have normal RB genes. For example: 1. Mutational activation of CDK4 and overexpression of cyclin D favor cell proliferation by facilitating RB phosphorylation and inactivation. Indeed, cyclin D is overexpressed in many tumors because of amplification or translocation of the cyclin D1 gene. 2. Mutational inactivation of genes encoding CDKIs also can drive the cell cycle by removing important brakes on cyclin/ CDK activity. 3. CDKN2A gene, which encodes the CDK inhibitor p16, is an extremely common target of deletion or mutational inactivation in human tumors.
- 36. FIRSTUP CONSULTANTS The loss of normal cell cycle control is central to malignant transformation and that at least one of the four key regulators of the cell cycle (p16, cyclin D, CDK4, RB) is mutated in most human cancers. Notably, in cancers caused by certain oncogenic viruses, this is achieved through direct targeting of RB by viral proteins. For example: 1. The human papillomavirus (HPV) E7 protein binds to the hypophosphorylated form of RB, preventing it from inhibiting the E2F transcription factors. 2. Thus, RB is functionally deleted, leading to uncontrolled growth.
- 40. FIRSTUP CONSULTANTS Conclusion: Within the limitations of this study, it seems that overexpression of Rb protein noted in oral cancer, with an increase in well and moderately differentiated tumors suggest a possible role of Rb in differentiation. The high expression of Rb in patients with combined habits of Paan chewing, smoking and alcohol consumption indicates that Rb pathway may be altered in habit-related oral malignancies. Keywords: Oral cancer, pre-neoplastic conditions, retinoblastoma protein, tumor suppressor gene, tumor suppressor proteins.
- 42. FIRSTUP CONSULTANTS The p53 protein is a transcription factor that thwarts neoplastic transformation by three interlocking mechanisms: 1. Activation of temporary cell cycle arrest (termed quiescence) 2. Induction of permanent cell cycle arrest (termed senescence) 3. Triggering of programmed cell death (termed apoptosis) p53 can be viewed as a central monitor of internal stress, directing the stressed cells toward one of these pathways.
- 44. FIRSTUP CONSULTANTS A variety of stresses trigger the p53 response pathways: 1. Anoxia 2. Inappropriate oncoprotein activity 3. DNA damage By managing the DNA damage response, p53 plays a central role in maintaining the integrity of the genome.
- 45. FIRSTUP CONSULTANTS In healthy cells, p53 has a short half-life (20 minutes) because of its association with MDM2(Mouse double minute 2 homolog), a protein that targets p53 for destruction. When the cell is stressed, by an assault on its DNA, “sensors” that include protein kinases such as ATM (Ataxia Telangiectasia Mutated) are activated. These activated sensors catalyse post translational modifications in p53 that release it from MDM2, increasing its half-life and enhancing its ability to drive the transcription of target genes. Hundreds of genes whose transcription is triggered by p53 have been found.
- 47. FIRSTUP CONSULTANTS These genes suppress neoplastic transformation by three mechanisms: 1. p53-mediated cell cycle arrest may be considered the primordial response to DNA damage. 2. p53-induced senescence is a form of permanent cell cycle arrest. 3. p53-induced apoptosis of cells with irreversible DNA damage is the ultimate protective mechanism against neoplastic transformation.
- 49. FIRSTUP CONSULTANTS 1. p53-mediated cell cycle arrest may be considered the primordial response to DNA damage. a) It occurs late in the G1 phase and is caused mainly by p53-dependent transcription of the CDKI gene p21. b) The p21 protein inhibits cyclin–CDK complexes and prevents phosphorylation of RB, thereby arresting cells in the G1 phase. c) Such a pause in cell cycling is welcome, because it gives the cells “breathing time” to repair DNA damage. d) If DNA damage is repaired successfully, p53 up regulates transcription of MDM2, leading to its own destruction and relief of the cell cycle block. e) If the damage cannot be repaired, the cell may enter p53- induced senescence or undergo p53-directed apoptosis.
- 50. FIRSTUP CONSULTANTS 2. p53-induced senescence is a form of permanent cell cycle arrest: a) It is characterized by specific changes in morphology and gene expression that differentiate it from quiescence or reversible cell cycle arrest. b) Senescence requires activation of p53 and/or Rb and expression of their mediators, such as the CDKIs. c) The mechanisms of senescence are unclear but seem to involve global chromatin changes, which drastically and permanently alter gene expression.
- 51. FIRSTUP CONSULTANTS 3. p53-induced apoptosis of cells with irreversible DNA damage is the ultimate protective mechanism against neoplastic transformation: a) It is mediated by up regulation of several pro-apoptotic genes, including BAX and PUMA.
- 53. FIRSTUP CONSULTANTS Confirming the importance of TP53 in controlling carcinogenesis, more than 70% of human cancers have a defect in this gene. Biallelic abnormalities of the TP53 gene are found in virtually every type of cancer like: 1. Carcinomas of the lung 2. Colon 3. Breast —the three leading causes of cancer deaths. In most cases, mutations affecting both TP53 alleles are acquired in somatic cells. In other tumors, such as certain sarcomas, the TP53 gene is intact but p53 function is lost because of amplification and overexpression of the MDM2 gene, which encodes a potent inhibitor of p53.
- 54. FIRSTUP CONSULTANTS As with RB, normal p53 also can be rendered non-functional by certain oncogenic DNA viruses. Proteins encoded by oncogenic HPVs, hepatitis B virus, and possibly Epstein-Barr virus can bind to normal p53 and nullify its protective function. Thus, DNA viruses can subvert two of the best understood tumor suppressors, RB and p53.
- 57. FIRSTUP CONSULTANTS Abstract. The retinoblastoma (Rb) and p53 genes play a fundamental role in cell cycle mechanisms, and their deregulation is related to many steps of oral cancer carcinogenesis. This study was conducted to evaluate the expression of the p53 and Rb proteins in malignant and pre-malignant oral cavity lesions. Excisional biopsy samples of oral cavities were collected from patients with suspected oral lesions. The samples were processed by immunohistochemistry. Seventy-one patients were studied (75% male; median age, 52 years; range, 24- 84). Of the samples studied, 59.4% were oral squamous cell carcinoma (OSCC) and 40.6% were pre-neoplastic lesions (leukoplakia and actinic cheilitis). OSCC presented higher expression of Rb protein compared to pre-malignant lesions. Pre-neoplastic lesions presented higher expression of p53 protein compared to OSCC lesions. Despite the small number of samples, the expression of these cell cycle biomarkers (p53 and Rb protein) in excisional biopsies suggests that molecular lesion assessment can determine pre-malignant lesions, and that its use may improve the clinical and surgical treatment of early lesions. Thus, p53 protein expression may be related to the early steps of carcinogenesis in OSCC. Finally, a higher Rb expression was also observed in malignant lesions.
- 59. FIRSTUP CONSULTANTS TGF-β, is a member of a family of dimeric growth factors that includes bone morphogenetic proteins and activins. In most normal epithelial, endothelial, and hematopoietic cells, TGF-β is a potent inhibitor of proliferation. It regulates cellular processes by binding to a complex composed of TGF-β receptors I and II. Dimerization of the receptor upon ligand binding leads to a cascade of events that result in the transcriptional activation of CDKIs with growth- suppressing activity, as well as repression of growth-promoting genes such as MYC, CDK2, CDK4, and those encoding cyclins A and E.
- 60. FIRSTUP CONSULTANTS In many forms of cancer, the growth-inhibiting effects of the TGF-β pathways are impaired by mutations affecting TGF-β signaling. These mutations may alter the type II TGF-β receptor that serve to transduce antiproliferative signals from the receptor to the nucleus. Mutations affecting the type II receptor are seen in cancers of: 1. Colon 2. Stomach 3. Endometrium
- 61. FIRSTUP CONSULTANTS The TGF-B cell signaling pathway plays a complex role in cancer development, progression, and metastasis. SMADs function in cell signal transduction from TGFB1 ligands to activate gene transcription. Binding of the TGFB1 ligand dimer to the TGF-beta receptor type-2 (TGFBR2) promotes dimerization of TGFBR2 with TGFBR1 and results in transphosphorylation of TGFBR1. The activated TGFBR1 activates R-SMADs (SMAD2 and SMAD3) via phosphorylation. SMAD2 and SMAD3 trimerize with SMAD4. The SMAD trimer enters the nucleus to activate gene transcription and promote cell growth and survival.
- 64. FIRSTUP CONSULTANTS The rare hereditary disease Adenomatous Polyposis Coli (APC) is characterized by the development of numerous adenomatous polyps in the colon that have a very high incidence of transformation into colonic cancers. They consistently show loss of a tumor suppressor gene called APC, which exerts antiproliferative effects in an unusual manner. It encodes a cytoplasmic protein whose dominant function is to regulate the intracellular levels of β-catenin, a protein with many functions and an important component of the so-called WNT signaling pathway that regulates cell proliferation. The name WNT is a portmanteau created from the name Wingless and the name Int-1. β-catenin binds to the cytoplasmic portion of E-cadherin and can also translocate to the nucleus and activate cell proliferation. Here the focus is on the latter function of this protein.
- 66. FIRSTUP CONSULTANTS WNT is a soluble factor that can induce cellular proliferation. It does so by binding to its receptor and transmitting signals that prevent the degradation of β-catenin, allowing it to translocate to the nucleus, where it acts as a transcriptional activator in conjunction with another molecule, called TCF(T-Cell Factor). In quiescent cells, which are not exposed to WNT, cytoplasmic β-catenin is degraded by a destruction complex, of which APC is an integral part. With loss of APC in malignant cells, β-catenin degradation is prevented, and the WNT signaling response is inappropriately activated in the absence of WNT. This leads to transcription of growth-promoting genes, such as cyclin D1 and MYC, as well as transcriptional regulators that repress E-cadherin expression and thus reduce contact inhibition.
- 68. FIRSTUP CONSULTANTS APC behaves as a typical tumor suppressor gene. Persons born with one mutant allele typically are found to have hundreds to thousands of adenomatous polyps in the colon by their teens or 20s; these polyps show loss of the other APC allele. Almost invariably, one or more polyps undergo malignant transformation. APC mutations are seen in 70% to 80% of sporadic colon cancers. Colonic cancers that have normal APC genes show activating mutations of β-catenin that render them refractory to the degrading action of APC.
- 71. REFERENCES
- 72. FIRSTUP CONSULTANTS 1. ROBBINS BASIC PATHOLOGY 9TH EDITION 2. RETINOBLASTOMA AND P53 PROTEIN EXPRESSION IN PRE-MALIGNANT ORAL LESIONS AND ORAL SQUAMOUS CELL CARCINOMA. Marília Gerhardt De Oliveira, Luciana Maria Pereira Ramalho, Leonilson Gaião, Daniel Humberto Pozza And Ramon Andrade De Mello; MOLECULAR MEDICINE REPORTS 6: 163-166, 2012. 3. THE EXPRESSION OF RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN IN ORAL CANCERS AND PRECANCERS: A CLINICOPATHOLOGICAL STUDY. Sunila Thomas, Anita Balan, and Prabha Balaram; Dent Res J (Isfahan). 2015 Jul-Aug; 12(4): 307–314.
- 73. THANK YOU