The Cell Cycle Regulation And Tumor Suppressor Genes Biology Essay

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1.0 Introduction

Cancer is the accumulation of several genetic alterations occurring in a cell that lead to uncontrolled growth of the cells. This genetic alterations can change the balance between proliferation and cell death programme mechanism (apoptosis) and cell transforming. Malignant transformation is included stages of initiation, promotion and progression. Usually, there is a long latent phase from the time of carcinogenic exposure to the stage of neoplastic transformation, in which the somatic cell is capable to proliferate though accumulating various genetic mutations that may consequence to a disease in exposed individual. Many factors showed to be linked with the oncogenic process, such as lifestyle, environment, infectious agent, host factors and inheritance. One of the preventable serious causes of cancer is infectious agents. viruses are the most common infectious agents that play a critical role in human cancer pathogenesis. Recent studies estimated that 17.8% of cancer cases are caused by infectious agents and 12.1% of these are due to viral infections. However our main interest point is DNA viruses which subvert the normal function of tumor suppression of cell growth and their contribution to cancer [1]. (see Figure1)

1.1 The cell cycle regulation

During the cell cycle, the DNA strands are copied followed by the DNA code transmission. Hence, the cell replication and division depend on four stages to produce genetically same daughter cells, namely the G1 (gap), S (synthesis), G2 and M (mitosis) phases of the cell cycle.

In the G1 phase, for the DNA duplication the cell builds up the cytoplasmic materials. DNA status checking occurs before cycle progression at the first stop (the R checkpoint) of the cell cycle. In case of any abnormality occurs in the genetic information cell cycle arrest takes place in case this not repaired. In the next phases G2 and S, DNA replicates and all materials are obtained for the cell duplication. In the cell cycle, last step is M phase, where the cell duplication takes place [3] . (see figure2).

Cell cycle progression is controlled by a group regulatory proteins known as cyclin-dependent kinases (CDKs) (see figure3). Besides, these enzymes appear in active in form of compounds with cyclins proteins. There is frequently interaction with CDK inhibitors and Proliferating Cell Nuclear Antigen (PCNA). Transitions of the cell cycle begin when the enzymatic activity of a specified kinase activates the proteins which are essential for progression from one phase of the cycle to the next. Moreover, every cell is programmed for particular functions and ends its life cycle throughout apoptosis (the genetic manage for removing improper or senescent cells) [4].

This understanding of the normal cell functions and regulation has appreciably contributed to our knowledge of different molecular mechanisms in human carcinogenesis. In this essay, I will give a brief description of HPV and (HHV-8/KSHV) roles, as the most important etiological agent of cervical cancer and Kaposi carcinoma , by describing the molecular mechanisms whereby these tumor viruses known to interfere with the regulation of the normal cell cycle and subverted for their benefit .

1.2 Tumor Suppressor Genes:

There are three types of genes that support tumor formation: genes that control the cell proliferation, genes that control the cell death or apoptosis programme, and genes necessary for damaged DNA repairing. Depending on how they influence each process, they grouped into two general categories:

tumor suppressor genes


Tumor suppressor genes are defined as the genes that encode proteins inhibit the tumors formation. Their usual role is to inhibit the cell growth, or work as the cell cycle brakes. Mutations in the tumor suppressor genes cause cancer development by inactivating their inhibitory function. These genes play a significant role in cells regulating when allowed to divide and increase in number. Once DNA damage in a cell is detected, a number of tumor suppressor genes stop the cell from multiplying till the damage is repaired. In addition, specific tumor suppressor genes could stimulate cells with DNA damage to commit or "cell suicide". When the tumor suppressor genes don't work correctly, the cells with damaged DNA maintain to divide and may accumulate more DNA damage that could ultimately lead to cancer cell formation [6]. This type of mutations are named loss-of-function mutations. As long as the cell includes one functional copy of a specified tumor suppressor gene (expressing sufficient protein to manage cell growth), that gene could inhibit the tumors formation. Inactivation of a tumor suppressor gene both copies is necessary before their role can eliminated. Thus, mutations in the tumor suppressor genes at level of an individual cell are recessive [7].

1.2.1 Retinoblastoma gene (Rb)

Rb gene was identified in retinoblastoma and it is the first tumor suppressor gene ever cloned. The Rb gene is located on chromosome 13 and encodes a nuclear protein that controls gene expression. Losing the function of the pRb pathway, leads to loss of the normal inhibitory of the cell cycle progression control [3]. Presence of Rb may prevent tumour formation by inducing cell differentiation, maintaining genomic stability, controlling cell cycle arrest and induce senescence as a response to oncogenic stress. Moreover, the absence of Rb has been linked with the increased of angiogenesis and metastasis. Interestingly, the presence of Rb has a pro-survival role because of its ability to inhibit cell death by apoptosis and autophagy. Thus, in some contexts this may be essential for early tumor cell survival[8]. (figure 4).

1.2.2 p53: a key tumor suppressor

P53 is inactivated in the majority of human tumors by loss-of-heterozygosity (LOH), and is therefore among the most important "brakes" on tumor formation. The p53 encodes a 53-kilodalton (kd) nuclear phosphoprotein which is usually present in very small quantities and has extremely short half life in the normal cells. The p53 gene is activated after DNA being damaged and its protein interacts with CDK cyclin inhibitors (p16, p27 and p21waf1cip1). This determined action leads to arrest the cell cycle at the point R, in the G1 phase, to let the DNA to repair (figure 5). If the recovery of DNA is successful, the p21 send a signal to CDK/cyclin compound to continue the cell cycle [7].

1.2.3 Genes encoding Cdk inhibitors are tumor suppressor genes p16

an inhibitor of Cdk4-cyclin D complexes, is lost in inherited melanoma. The loss of p16 can lead to increased the activity of Cdk4-cyclin D, inactivation and phosphorylation of Rb, consequence in E2F activation and transcription of cyclin.

1.2.4 pVHL-multifunctional tumor suppressor protein

The von Hippel-Lindau (VHL) tumor-suppressor gene is commonly inactivated in VHL disease and in renal cell carcinoma sporadic cases. VHL protein (pVHL) acts as an E3 ubiquitin ligase compound that targets several cellular proteins for proteasomal degradation [10].

2.0 DNA viruses:

More than three decades studies conducted evident that several viruses play a key role in the progression of human malignancy. A group of oncogenic DNA viruses with diverse structures, different replication strategies and genome organization are presented in human population, these elements are considered to be infectious agents of cancer. This includes the Human papillomaviruses(HPVs), Kaposi's sarcoma associated herpesvirus (KSHV, also known as HHV-8), Epstein Barr virus (EBV) and Human polyomaviruses. Universally, 20% of all different cancers are correlated to infectious agents [11] (figure 6).

DNA viruses lytic cycle totally dependent on their capability to affect the replicative machinery of their host cell. therefore, like tumor cells, DNA tumor viruses have to change the mechanisms that separate cellular replication from various intracellular and extracellular factors, which are normally responsible to control it. accordingly, tumor cells and DNA viruses subvert a lot of the similar cellular checkpoints. For instance, the Rb/p53 tumor suppressor genes checkpoints are inactivated in tumor cells through mutations. Viruses can cause cancer by the integration of viral DNA into the host normal cell DNA and this can result in new genes or muddles up the host old ones [11].

Its believed that DNA viruses can cause cancer because of following reasons:

Virus can carry in a new gene/protein , which is a gene or an oncogene that codes a tumor suppressor inhibitor (which can cause cervical cancer) and makes 2 proteins that can inhibit the p53 and Rb tumor suppressors (figure7).

b. Virus DNA can destroy a host gene : by the integration into the host DNA in the middle of normal gene and inactivate it, which leads to knock out a tumor suppressor.

c. Virus DNA can carry in a new regulatory sequence : by the integration into DNA close to a normal host gene and give silencer or an enhancer that alters that host gene expression, which can switch on a proto-oncogene [13].

2.1 Human papillomaviruses (HPV)

HPV is non-enveloped, small and double stranded DNA tumor virus. It is a member of the papovavirus family. It can cause different epithelial lesions that can be transformed from benign lesions such as mild cervical dysplasia, recurrent respiratory papillomatosis and anogenital warts, to pre-malignant and malignant lesions. Morethan 100 types of HPV have been recognized, but only some strains have established sufficient confirmation to be elected as definitive human carcinogens. These strains include HPV-16 and HPV-18, which consider as high risk types. Globally, HPV-16 and HPV-18 are the reason for about 70% of anal and cervical cancers. On the other hand, Low risk types are HPV-6 and HPV-11, accounting for about 90% of genital warts [15,16]. HPV infects well differentiated keratinocytes of the stratified squamous epithelium at the basal epithelial layer. It randomly integrates into the host genome and encodes for 6 non structural viral regulatory proteins (E1, E2, E4, E5, E6 and E7) from the viral genome early region and two mstructural viral capsid proteins from the late region(L1 and L2) [16].

2.1.1 Transformation and oncogenesis

HPV primary infection occurs in the basal stem cells of the epithelium. Then it traverses upward and replicates in the incurably differentiated keratinocytes and is shed from the layer corneum [17]. HPV lacks a polymerase gene, so its replication depends on the stimulation of cellular DNA synthesis in these cells. In most cervical carcinomas, due to the integration of HPV into the host genome leads to loss of E2 viral gene expression, which is known to be the transcriptional repressor of E6 and E7 gene expression. Consequently, this leads to increase the expression of E6 and E7 viral oncoproteins [17].

2.1.2 Deregulation of the cell cycle by E6 and E7

Currently, it is recognized that certain HPV genes could influence cell cycle regulation to help viral persistence and replication. The E6 and E7 proteins of HPV bind to the cell cycle regulatory proteins and then interfere with the G1/S and G2/M cell cycle checkpoints. In-vivo, many chromosome abnormalities have been recognized in low grade cervical lesions were infected with high risk HPVs. This associates with the in vitro observations that HPV16 (E6 and E7) can alter cell cycle control and can induce chromosome abnormalities in fibroblasts and normal epithelial keratinocytes. Additionally, the high-risk HPV proteins can:

up- regulate the cyclins A and B expression associated with immortalization.

2) up-regulate the expression of cyclin E, recently shown to induce the genetic instability.

3) abrogate the expression of cyclin D1, which is significant in the Rb pathway [18].

High risk HPV strains proteins E6 and E7 have a strong transforming abilities. In vitro, it has been shown that E6 and E7 viral proteins can induce skin tumors in transgenic animals and immortalize cells [19]. In addition, these proteins causes dysregulation of the cell growth by targeting the tumor suppressors.

E6 viral protein binds p53 and stimulates its degradation, while E7 viral protein binds retinoblastoma (Rb) family members. These consequence in cell cycle dysregulation and inhibition of apoptosis. Furthermore, E6 combines to a ubiquitin ligase (E6- AP) forming a complex binds p53 and consequence in ubiquitination and proteosomal destruction of the protein. Moreover, E6 could induce the telomerase activity and cells immortalization. On the other hand, the E7 protein obstructs Rb function and allows cells to progress to S phase. Also E7 connects the hypophosphorylated form of Rb and stops it from binding to the E2F transcription factor. Free E2F promote the genes expression that essential for cell DNA synthesis, thus pushing the cell into the cell cycle [20]. In addition, E7 stimulates the activity of cyclin-A and cyclin E dependent kinase and inactivates p27/KIP1and p21/WAF1, the cyclindependent kinase inhibitors. Abnormal centriole synthesis and aneuploidy can be promoted by E7early in the oncogenic process.

Additionally, E6 and E7 expressing cells have less ability to sustain genomic integrity. The E7 oncoprotein function as a mitotic mutator and stimulates multiple types of mitotic abnormalities, involving anaphase bridges, lagging chromosomes or unaligned and most particularly multipolar mitoses[21], which are the histopathological characteristics of high risk HPV linked cervical lesions and cancer and caused by high risk HPV ability to separate centrosome duplication from cells division cycle [43]. Thus, HPV E6 and E7 oncoproteins mechanistically have been contributed to initiation and cervical cancer progression (see figure8).

Moreover, the studies of cervical cancer showed that the HPV E6 and E7 protein joined with pRb is deactivated, and eliminates the negative inhibition of P16INK4A protein expression, consequence in P16INK4A over-expression. The P16INK4A gene is a significant member of the P53-Rb system, and its P16INK4A protein product can stop the cell from entering S-phase from the G1-phase, and also suppress the cell proliferation, during inhibition of retinoblastoma (pRb) phosphorylation[22].

Furthermore, E6 oncoprotein targets MAGI- 1tumor suppressor and SAP97/hDlg for degradation, which is a human homolog of Drosophila discs large [24], MAGI-1, 2 and 3 [25]. In addition to the non MAGUKs MUPP1 (1) and hScrib, which is a human homolog of the Drosophila scribble [26]. The HPV18-E6 proteins target a group of membrane associated guanylate kinase (MAGUK). E6 can identify and compete for PDZs of cellular tumor suppressors in the cell transformation process and oncogenesis. A short peptide of E6 at the C-terminus interacts particularly with the domains of PDZ of these tumor suppressors and may leads to differention, dystruction of organisation and of chromosomal integrity of HPV infected cells. Accordingly, may contribute to morphological transformation and induction of hyperplasia, which is a assets unique to high risk HPVs that are linked with cervical cancer .

Finally the characteristic of HPV to induce carcinogenesis is inactivation of p53 by E6 and Rb by E7 of high risk HPV genotypes. The abnormal function of these genes and the resulting genomic instability in addition to additive effects of one or more cofactors such as tobacco, dietary agents, steroids and UV and X radiation directs the affected cells to preferential growth, which characterize the uncontrolled and progressive growth in cancer as it is shown in figure 9.

2.2 Human herpes virus 8 (HHV-8) / Kaposi sarcoma associate herpesvirus (KSHV)

Human herpes virus 8 (HHV-8), also known as Kaposi's Sarcoma associated Herpes Virus (KHSV), is a double stranded DNA virus classified in the Rhadinovirus genus, which belongs to the γ-2 herpesviridae sub-family. This virus is the etiological reason of Kaposi's sarcoma (KS). It transmit sexually and remains latent with the opportunity of reactivation in the immunocompromised individuals. It is a multifocal angioproliferative illness, usually appeared as a cutaneous lesion. It is also linked with other proliferative diseases such as Multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL) [28]. (see figure 10 below).

KSHV virus can establish lifetime persistence in the infected host following primary infection. There are four types of KS:

First: is the classic type, that exists in Mediterranean persons.

Second: composed in the association among KS and AIDS, where patients usually have disseminated KS.

Third: is the endemic type.

Fourth: is the iatrogenic form, that arise in individuals taking immunosuppressive therapy. KHSV/HHV-8 starts latent infection in the infected host, continuing episomally in B lymphocytes [30].

2.2.1 Cell cycle regulation by KSHV

KSHV has two phases; a latent and a lytic phase that encode antigens that shown to be able to block the cell cycle regulatory checkpoints, apoptosis control machinery importantly, the immune response regulatory mechanisms and apoptosis control machinery [33]. Hence, this cellular regulatory networks inhibition shows to be a defensive means which permits the virus to get away from antiviral innate immune response. Though, due to the innate immune system overlapping nature and tumor suppressor different pathways, these regulatory networks inhibition can consequence in un-regulated cell proliferation and can contribute to virus induced tumorigenesis. throughout the latent phase, it avoids the immune system, persisting in the infected host cell including a restricted genes expression.

2.2.2 Latent KSHV Infection phase

There are more than 90 open reading frames (ORFs) recognized in KSHV genome, but during the latency a small number of genomes are expressed, including LANA (Latency-associated nuclear antigen) which is encoded by ORF73, vCyclin, vFLIP/K13, K12/Kaposin and an miRNA cluster. V-cyclin stimulates progression of the cell cycle, vFLIP and vIRF3 mediate pro-survival signalling system [31], and kaposins stimulate the expression of the cytokine and cell growth [32].

LANA has shown to play a key role in deregulating a variety of cellular functions including:

Viral episome maintenance.

degradation of tumor suppressors (p53 and pRb).

Telomerase reverse-transcriptase promoter transactivation,

chromosome instability promotion in KSHV infected B cells.

intracellular domain of Notch accumulation in KSHV mediated tumorigenesis [33].

LANA also can inhibit the RTA expression, another significant viral encoded transcriptional activator essential for regulating the switch from the latent to the lytic phase consequently, the latency maintenance [34]. Importantly, LANA binds the viral episomal DNA to the infected host chromosomes, which assists in the competent division of the viral DNA in the new daughter cells. Hence, LANA expression disruptions direct to episomal copies reduction in, suggesting the significance of LANA in KSHV mediated pathogenesis. Interestingly, LANA and RTA both have been shown to interact with HAT protein CBP signifying their functions in chromatin modification [34].

Additionally, LANA has the ability to interfere with the p53 functional activity and block apoptosis [35] .LANA and EBNA3C alter p53 function by inhibiting its transcriptional activity and also LMP1can block p53 mediated apoptosis during the A20 gene induction [35]. LANA stimulates the β-catenin pathway by the intracellular redistribution regulation of GSK-3β. Besides, LANA increases GSK-3β nuclear accumulation and ironically reduces nuclear GSK-3β activity during a stable interaction [36].

As well LANA can also interact with pRb and increase E2F dependent transactivation activity, demonstrating that LANA by targeting pRb-E2F transcriptional regulatory pathway contributes to the KSHV induced oncogenesis [33].

Furthermore, the KSHV also encoded v-cyclin, which is a homolog of cyclin D1 [37]. Like its cellular complements, v-cyclin binds and activates the kinase partner(CDK6) results in increasing the pRb phophorylation and facilitating transition of the G1-S. Moreover, the v-cyclin/ CDK6 association induces the phosphorylation of a wide variety of significant cellular substrates involving histone H1, cdc25A, p27KIP1 and components of the DNA replication machinery. The v-cyclin and CDK6 phosphorylation of the p27KIP1 stimulates its proteasome mediated degradation and also increases endogenous cyclin- CDK complexes activation (see figure 11) [37].

Interestingly, KSHV encoded LANA has been shown to assists pVHL degradation along with p53 by recruitment of EC5S ubiquitin complex [38]. VHL protein (pVHL) works as an E3 ubiquitin ligase complex, that targets several cellular proteins for proteasomal degradation.65 HIF-α (hypoxia inducible transcription factor) is the best characterized cellular target, whose activity is controlled by pVHL [39]. Now it is obvious that loss of the pVHL can consequence in the activation of many cellular pathways that are associated with tumor initiations and progression. Furthermore, pVHL form a constant complex with p53 and inhibits Mdm2-mediated ubiquitination/degradation. Thus, Stabilization of the p53 results in initiation of p53 transactivation functions.

2.2.3 Lytic KSHV Infection phase

During lytic replication (see figure 11), KSHV produces a range of immunomodulatory proteins with paracrine properties. KSHV contains numerous gene products with the transforming properties. Viral G-protein coupled receptor (vGPCR)is the most significant between this gene products, which is an IL-8 receptor analog and vIL-6 that alters cell growth.

The K1 protein induces VEGF expression, while the v-Bcl-2 and v-cyclin proteins can inhibit apoptosis. They stimulate pathways controlling the cellular growth, angiogenesis and inhibition of apoptosis such as the PI3K MAPK family Jak/STAT and Nuclear Factor-κB (NF-κB) signaling pathway [40]. In vitro, these proteins have shown transforming properties and also in transgenic mice.

Furthermore, there are two KSHV encoded constitutive signaling membrane proteins, variable ITAM containing protein (VIP) and (LAMP) latency associated membrane protein, that play a part in KSHV associated malignancies. Additionally, K1 encodes VIP protein that transforms rodent fibroblasts, which, after injected into nude mice, induces multiple and distributed tumours. LAMP, which is encoded by the K15 ORF, shows latent mitogenic and survival signaling by the activation of Src-family kinases and NF-κB and might promote the cell survival through interaction with Bcl- 2 associated anti-apoptotic protein HAX-1 [23]. Finally, KSHV also includes many immune evasion genes such as, vIRFs, Orf45, MIR1, MIR2, and complement control protein homolog (CCPH). The KSHV MIR1,MIR2, K3 and K5 proteins can downregulate the MHC I expression, accordingly inhibiting the viral antigen presentations. While the vIRFs and Orf45 inhibit the infected host interferon response, whilst CCPH inhibits the complement-mediated lysis of the infected cells. Together, all these immune evasion proteins ensure the viral life-long persistence in the host, and consequently contribute to KSHV associated pathogenesis [41].

KSHV infection in the universal population rarely causes KS. This emphasize the continuation of cofactors, such as HIV or drug-induced immunosuppression, that are essential for the virus to induce a tumor.

3.0 Conclusion

Tumorigenesis is considered to be a multistep process and infection with the DNA tumor viruses can substitute for one or many of the mutational events that result in complete transformation, neoplasia and metastasis. As described above, HPV and KSHV encode a diverse range of viral genes that help contribute to neoplastic process. These viruses have evolved strategic means of deregulating and perturbing normal cellular pathways that would otherwise lead to apoptosis, activation of the host immune system and cell growth arrest. The rest of the genes encoded by these viruses have roles in viral replication, packaging, entry and immune evasion.