Which of the following cellular events occur in the g1 phase of the cell division cycle?

Deregulation of G1 Restriction Point Control in Cancer

In G1 phase, cells make the decision to either progress through the restriction point and enter S phase or enter G0. These decisions are based on extracellular signals that the cell receives and on the integrity of signaling machinery that detects these signals. Deregulation of G1 progression is a frequent occurrence in cancer. This can occur through mutations or deregulated expression of CDKs, cyclins, or CKIs. Loss- or gain-of-function mutations in upstream regulators of the CDK kinases also occur in cancer. In this section, we discuss some alterations found in cell cycle regulators in cancer.

Cyclin D–dependent kinases are a primary point of control for the progression through G1 phase and are linked to cancer progression. Cyclin D1 overexpression is a hallmark of breast and esophageal cancers (58). In many cases this up-regulation is due to cyclin D1 gene amplifications, but can also result from increased transcription (58). In addition to gene expression alterations, decreased cyclin D1 proteolysis is implicated in deregulated cyclin D/CDK4 activity in breast and esophageal cancers. Cyclin D1 overexpression also occurs as a consequence of chromosomal translocations. Amplifications encompassing the CDK4 and CDK2 genes have been reported in large B-cell lymphomas, lung tumors, and cervical carcinomas. Downstream targets of cyclin D/CDK4/6 kinases, Rb proteins, are also targeted in cancer. Mutations and deletions in the Rb gene are common events in tumors; inactivation of Rb alleviates a cell need for CDK4/6 kinase and thus relieves some cellular dependence on growth factor signals (59).

As one might anticipate, Cip/Kip inhibitors can also function as tumor suppressor proteins in mouse model systems and consistent with this work, their expression is deregulated in human cancers. p53, the main transcriptional regulator of p21Cip1 is often lost or mutated during tumorigenesis. Reduced p27Kip1 levels alone or together with increased cyclin E expression are associated with poor prognosis in breast and ovarian carcinomas. Inactivation of p16Ink4a occurs frequently in lung, bladder, and breast carcinomas, as well as leukemia (reviewed in [24]).

In addition to alterations in the expression and integrity of cell cycle genes in cancers, attenuation of their regulatory pathways also occurs. These include signaling pathways (Ras), transcription factors (myc), and components of ubiquitin ligases. Skp2, the specificity component of the SCF ligase for p27Kip1, is up-regulated in variety of tumors, including colon, lung, breast, prostate, and lymphoma (54), where it decreases p27Kip1. Another F-box protein, Fbw7, which regulates degradation of cyclin E, is mutated in ovarian and breast cancers.

Mutations and deregulation of the expression of regulators of mitosis are also observed in human malignancy. Increased accumulation of Cdc20 (APC/C) is observed in lung and gastric tumor cell lines. Mutations in PLK1 are found in human cancer cell lines and its attenuated expression is observed in colorectal, endometrial, and breast carcinomas.

Chromosome Dynamics During the Cell Cycle

The G1, S, and G2 phases are often referred to as interphase, and the M phase is the mitotic phase. During interphase, chromosomes are replicated, and during mitosis they become highly condensed and then are separated and distributed to the two daughter nuclei. The highly condensed chromosomes in a dividing cell are known as mitotic chromosomes. During the portion of the cell cycle when the cell is not dividing, the chromosomes are extended and much of their chromatin exists as long, thin tangled threads in the nucleus (Fig. 5.2).

As the cell enters M phase, the nuclear membrane is disassembled, and sister chromatids condense into compact structures that remain bound together. The centromeres of the condensed sister chromatids bind microtubules, which form the mitotic spindle. The spindle organizes the sister chromatid pair at the center of the cell, which is known as the kinetochore. Subsequently, each sister chromatid moves to the opposite pole on the spindle. The set of chromosomes at each pole become encapsulated by a nuclear membrane, and the cell divides into two daughter cells. Therefore, the chromosome structure is a dynamic structure that can be condensed and extended throughout the cell cycle.

Condensed human mitotic chromosomes have been studied under the microscope for many years. The display of the chromosome set of an individual, lined up from the largest to the smallest, is called a karyotype (Fig. 5.3). Therefore, a karyotype is referred to as the microscope images of metaphase chromosomes when the sister chromatids are maximally condensed but have not yet separated.

Deregulation of G1 Restriction Point Control in Cancer

In G1 phase, cells make the decision to either progress through the restriction point and enter S phase or enter G0. These decisions are based on extracellular signals that the cell receives and on the integrity of signaling machinery that detects these signals. Deregulation of G1 progression is a frequent occurrence in cancer, through mutations or deregulated expression of CDKs, cyclins, or CKIs. Loss- or gain-of-function mutations in upstream regulators of the CDK kinases also occur in cancer. In this section, we discuss some alterations found in cell cycle regulators in cancer.

Cyclin D–dependent kinases are a primary point of control for the progression through G1 phase and are linked to cancer progression. Cyclin D1 overexpression is a hallmark of breast and esophageal cancers.75 In many cases this upregulation is due to cyclin D1 gene amplifications, but it can also result from increased transcription.75 In addition to gene expression alterations, decreased cyclin D1 proteolysis is implicated in deregulated cyclin D/CDK4 activity in breast and esophageal cancers. Cyclin D1 overexpression also occurs as a consequence of chromosomal translocations. Amplifications encompassing the CDK4 and CDK2 genes have been reported in large B-cell lymphomas, lung tumors, and cervical carcinomas. Downstream targets of cyclin D/CDK4/6 kinases, Rb proteins, are also targeted in cancer. Mutations and deletions in the Rb gene are common events in tumors; inactivation of Rb alleviates a cell need for CDK4/6 kinase and thus relieves some cellular dependence on growth factor signals.76

As one might anticipate, Cip/Kip inhibitors can also function as tumor suppressor proteins in mouse model systems, and, consistent with this work, their expression is deregulated in human cancers. p53, the main transcriptional regulator of p21Cip1, is often lost or mutated during tumorigenesis. Reduced p27Kip1 levels alone or together with increased cyclin E expression are associated with poor prognosis in breast and ovarian carcinomas. Inactivation of p16Ink4a occurs frequently in lung, bladder, and breast carcinomas, as well as leukemia (reviewed in Ref. 24).

In addition to alterations in the expression and integrity of cell cycle genes in cancers, attenuation of their regulatory pathways also occurs. These include signaling pathways (Ras), transcription factors (myc), and components of ubiquitin ligases. Skp2, the specificity component of the SCF ligase for p27Kip1, is upregulated in a variety of tumors, including colon, lung, breast, prostate, and lymphoma,70 where it decreases p27Kip1. Another F-box protein, Fbw7, which regulates degradation of cyclin E, is mutated in ovarian and breast cancers.

Altered functionality of cyclin D1 ubiquitin ligase can lead to increased cyclin D1 expression and ultimately to tumorigenesis. Cyclin ubiquitination requires both FBXO4 and a specificity co-factor, αB-crystallin.77 αB-crystallin expression is lost or downregulated in breast cancer and melanoma cell lines, which correlates with decreased cyclin D1 proteolysis.78,79 Primary esophageal carcinomas, which are known to frequently overexpress cyclin D1, exhibit hemizygous, missense mutations of FBXO4.80

Mutations and deregulation of the expression of regulators of mitosis are also observed in human malignancy. Increased accumulation of Cdc20 (APC/C) is observed in lung and gastric tumor cell lines. Mutations in PLK1 are found in human cancer cell lines, and its attenuated expression is observed in colorectal, endometrial, and breast carcinomas.

The S Period

During the G1 period, each cell contains one diploid copy of the genome. At the end of this period the cell enters the S period, during which the cell gradually increases in size, doubling its total mass. During S, each homologue replicates to become a two-part chromosome consisting of two sister chromatids. These two sister chromatids are held together at the centromere, a specialized region near the center of the chromosome. Individual chromosomal segments have their own characteristic replication time during this period of about 9 hours. By the end of this period, the cell contains two full copies of its chromosomal material. In other words, at this point the cell has become, temporarily, doubly diploid.

Cyclin E

Cyclin E regulates G1 phase progression and entry into S phase. There are two different proteins, cyclin E1 and cyclin E2, that are coded by two different genes with 47% homology. Several splice variants of cyclin E1 not present in normal cells have been identified and these seem to stimulate cells to progress through the cell cycles more than wild-type cyclin E1. The most frequently used determination method for cyclin E1 is IHC, although western blot allows for assessment of total as well as isoform-specific expression. Cyclin E mRNA can also be measured by reverse transcription polymerase chain reaction (RT-PCR).

Elevated levels of both cyclin Es are more frequently found in ER negative tumors. Many retrospective studies have demonstrated an association between high levels of cyclin E and an increased risk of breast cancer-related death, although this finding has not been seen across all the trials. Furthermore, lack of standardization regarding evaluation methods and scoring systems limits its use as a prognostic factor. There is even less evidence for the use of cyclin E as a predictive tool [95].

G1/S Checkpoint

Cells in the G1-phase of the cell cycle become committed to enter the S-phase at a stage referred to as the Restriction point (R) in mammalian cells and Start in budding yeast. The G1/S checkpoint serves to prevent cells from entering S-phase in the presence of DNA damage and functions to inhibit the initiation of replication (Figure 2). In the case of double-stranded breaks (DSBs), caused by ionizing radiation or radiomimetic agents, the ataxia telangiectesia mutated (ATM) kinase is activated and phosphorylates many downstream effectors, notably p53 and Chk2. Activation of these signal transduction pathways serves to initiate (Chk2) and maintain (p53) the G1/S arrest. The initiation of G1/S checkpoint requires Chk1- or Chk2-dependent phosphorylation of the Cdc25A phosphatase, leading to its nuclear exclusion and ubiquitin-mediated degradation. The absence of Cdc25A prevents the dephosphorylation of Cdk2 at the inhibitory phosphorylation sites and thus enforces the inactivation of Cdk2/cyclin complexes. Without active Cdk2, the Cdc45 protein, which is one of the key components of the pre-replication complex, is not phosphorylated and is unable to be loaded onto chromatin to initiate replication. This results in a block to the initiation of DNA synthesis. The phosphorylation-regulated pathway is implemented rapidly in response to DNA damage but is relatively transient and delays G1/S transition for just a few hours. Maintenance of the G1/S arrest requires DNA damage-induced activation of p53, which stimulates the expression of p21/Cip1/WAF1, an inhibitor of Cdk2/cyclin complexes. Since upregulation of p21Cip1 involves transcription and new protein synthesis, this p53-mediated G1 arrest is a slower response that complements the transient acute inhibition of Cdk2 through Cdc25A degradation.

7.01.5.1.1 The G1/S transition and the increase of activity of cyclin-dependent kinases

Cells entering the G1 phase will actively prepare to divide (newly synthesized proteins are produced, the cell size increases), until a certain point called the G1 checkpoint. This crucial checkpoint is controlled by the retinoblastoma tumor suppressor gene product (Rb), which is a transcriptional regulator. This protein imposes a block on G1 progression that is released by its phosphorylation by Cdk4/cyclin D1, Cdk6/cyclin D3, and Cdk2/cyclin E. Hyperphosphorylated Rb (pRb) dissociates from the transcription factors of the E2F-DP family (principally E2F1, 2, and 3) making these proteins available to direct the expression of proteins essential for DNA synthesis such as cyclin A, dihydrofolate reductase, thymidine kinase, thymidylate synthase, or DNA polymerase-α. The activity of the CDKs is tightly regulated by endogenous inhibitors of Cdk/cyclin complexes. Thus, the p21cip1, p27kip1, and p57kip2 gene products can bind and inhibit all Cdk/cyclin D, E, and A complexes, and the products of the INK4 gene (p16Ink4a, p15Ink4b, p18Ink4c, and p16Ink4d) have the ability to interact specifically with CDK4 and 6.

The G1 checkpoint is often deficient in human tumors, often due to deregulation or absence of the Rb protein. Although germline mutations in the RB gene cause the highly penetrant hereditary retinoblastoma,84 the frequency of RB mutation is low among the sporadic cancers; however, it has been reported in osteosarcomas, small cell lung carcinomas, and breast carcinomas. Rb protein inactivation, found in a wide variety of human cancers,85 may be the result of three possible causes. First, the Rb protein can be sequestered from its physiological partners, when bound to viral oncoproteins, such as the SV40 T antigen, the adenovirus E1A protein, or the papilloma E7 protein.86 These events are frequently observed in human cervical tumors. The second cause, and probably the most common one, is the loss of Rb function through permanent hyperphosphorylation, leading to accumulation of active E2F factors. This can occur by deregulated expression of cyclin D or CDK4, as a result of amplification or translocation of the respective genes. For example, CDK4 is amplified in gliomas and sarcomas.87 Alternatively, point mutations abrogating p16Ink4a binding have also been identified in CDK4.88 Third, CKI genes such as the INK4 gene are often deleted 89 or silenced by hypermethylation of the gene promoter90 in human tumors.

Key observations made in different biological systems have also identified Rb as an important player in cell fate determination (i.e., the differentiation process), by inducing apoptosis. This can be accomplished by two different mechanisms: (1) through regulation of apoptosis either in a E2F1, p19ARF (the sixth and last product of the INK4A/ARF locus) and p53-dependent fashion,91,91–95 or (2) in a E2F1-independent manner, through c-Jun N-terminal kinase (JNK), nuclear c-Abl, and p84N5.96–100 Because for a cell to become tumorigenic, it has to turn out to be resistant to apoptosis (see Section 7.01.5.3) and acquire properties leading to a strong blockade of the cell death machinery.

Introduction

Microtubules are an important part of a cell’s cytoskeleton. Microtubules are responsible for maintaining a cell’s shape, transporting organelles within the cytoplasm, and, in the case of cilia or flagella, moving the whole cell. Microtubules also play a critical role in the cell cycle by forming the mitotic spindle. All of these important cellular functions depend on polymerization of alpha (α) and beta (β) tubulin protein dimers into a microtubule. The organelle responsible for nucleating the polymerization of microtubules is called a centrosome.

Centrosomes are made up of two centrioles surrounded by a mass of pericentriolar material (PCM). Centrioles are cylindrical structures composed of multiple sets of three short microtubules organized into a pinwheel-like pattern. The two centrioles are usually oriented at right angles to one another. The PCM is a matrix of protein and fibrous material. Nucleation of microtubule assembly occurs within the PCM. Microtubule growth is “seeded” by ring structures composed of the protein gamma tubulin (γ tubulin). γ tubulin rings are believed to attach to the fibers within the PCM.

A cell in the G1 phase of the cell cycle has a single centrosome containing a pair of centrioles. By M phase, however, a cell needs two centrosomes, each with its own pair of centrioles, which will function as the spindle poles required to build a mitotic spindle. This means that the centrioles must replicate as part of the cell cycle. Centriole replication begins at the G1/S phase transition. Replication is semiconservative with each centrosome containing one centriole from the original parent centrosome, and one newly formed centriole. Replicated centrosomes remain together until the cell enters prophase of mitosis.

Compare and contrast the following cell biology terms: centrosome, basal body, microtubule organizing center (MTOC).

Look up images of centrioles and centrosomes to gain a better understanding of the organization of this organelle.

Identify the spindle poles/centrosomes in images of mitotic spindles.

Describe another example of semiconservative replication in biology.

Describe the cellular changes associated with prophase of mitosis. What role do centrosomes play in prophase?

How does a mitotic cell restore the normal ratio of “one centrosome per cell?”

3.3 Gene ablation strategies for functional characterization in G. lamblia

Giardia trophozoites in the G1 phase of the cell cycle are tetraploids cells, with each nucleus containing two genome copies. Hence, functional characterization by ablation of any given gene or protein product requires targeting of four distinct loci. In the absence of haploid gametes and a sexual cycle, gene disruption in G. lamblia poses a challenge. Approaches to reduce or even abolish gene expression or mRNA translation based on antisense RNA and morpholinos, respectively, were shown to have limited efficacy (Carpenter and Cande, 2009; Rivero et al., 2010a).

By using a combination of sequential recombination and Cre/loxP-mediated antibiotic resistance gene excision (Wampfler et al., 2014), the first G. lamblia knock-out line was produced, carrying four non-functional cwp1 loci (Ebneter et al., 2016). A less time-consuming procedure for gene disruption in G. lamblia, based on the Streptococcus pyogenes class II CRISPR-Cas9 system, was recently developed and tested. Despite correct nuclear targeting of the Cas9 endonuclease concomitant with in vivo sgRNA expression, target gene expression could be significantly decreased but not abolished (Lin et al., 2019). A variant of this system based on a catalytically-inactive Cas9 as a transcription repressor to displace RNA polymerase II complexes yielded similar results (McInally et al., 2019). These data suggest that current CRISPR-Cas9 set-ups are not yet sufficient to obtain complete gene ablation in G. lamblia. Further efforts are therefore necessary to harness the full power of the CRISPR-Cas9 tool to perform genomic manipulations in this parasite.

Interphase

Interphase is subdivided into three phases: G1 (gap) phase, when the cell prepares to synthesize DNA; S (synthetic) phase, when DNA is replicated; and G2 phase, when the cell prepares for the mitotic event (see Fig. 3.8).

G1 phase: At the conclusion of mitosis, the newly formed daughter cells enter the G1 phase of the cell cycle, a stage characterized by the synthesis of the regulatory proteins necessary for DNA replication, the restoration of the nucleoli and of the original cell volume of the daughter cell, and the initiation of centriole duplication.

S phase: The S phase is the synthetic phase where the genome is duplicated. At this point, the cell's normal complement of DNA has doubled from the normal (2n) to (4n) in preparation for the mitotic event.

G2 phase: The interval between the end of DNA synthesis and the beginning of mitosis is known as the gap 2 phase; during this phase, the RNA, tubulin, and additional proteins required for cell division are synthesized. Additionally, adenosine triphosphate (ATP) reserves are increased, and the newly synthesized DNA is checked for possible errors and, if present, corrected.

CLINICAL CONSIDERATIONS

Cancer chemotherapy has been enhanced by a more complete understanding of the cell cycle and mitosis. Certain drugs can be employed at specific times to arrest cell proliferation by disrupting certain stages of the cell cycle. Vincristine disrupts the mitotic spindle arresting the cell in mitosis. Colchicine, a plant alkaloid, is used to produce the same effect and is used for individual chromosome studies and for karyotyping. Methotrexate, a drug that inhibits purine synthesis, and 5-fluorouracil, a drug that inhibits pyrimidine synthesis, act during the S phase of the cell cycle, preventing cell division, and are used in chemotherapy treatment.