Cell Proliferation and Cell Cycle

** Definitions, Vs Differentiation


Definition


Cell proliferation refers to the processes that result in an increase in the number of cells.

As such, it's a fundamental process among living organisms that is necessary for general development (embryonic development, organ growth, and development as well as various physiological processes).

The increase in the number of cells occurs through a sequence of steps that constitute the mitotic cycle, also known as generation cycle, cell cycle, or proliferative cycle.




Given that cell proliferation involves an increase in the number of cells, the number of cells present during this process is measured as a function of time.

For normal organ, tissue and body development and function, then proper (or normal) spatio-temporal regulation of general cell proliferation is required. In the event of abnormal regulation, over-proliferation may occur resulting in the accumulation of an abnormal number of cells. 

 

* While cell proliferation is essential for normal growth and development, it's worth noting that cells only proliferate as needed. For this reason, some of the somatic cells in tissue do not proliferate until it's necessary.

 


Mechanisms that Regulate Cell Proliferation


Under normal circumstances, cell proliferation will occur through the four stages of cell cycle that includes G1 phase, S phase, G2 phase, and the M phase. Before looking at these steps in detail, it's important to understand some of the controls that regulate cell proliferation. 

A number of environmental factors (internal environment) contribute to and regulate cell proliferation. This includes such factors as nutrients, temperature levels, pH, and oxygen among others. In turn, these factors contribute to the mechanisms controlling the rate of cell proliferation. 


Positive and Negative Controls


In multicellular eukaryotes, it's the balance between positive and negative controls that regulate the rate of cell proliferation. A good example of this is cell adhesion. In epithelial tissues, for instance, close cell adhesion negatively controls proliferation (it's reduced). 

In the event of an injury that results in a loss of adhesion, negative adhesion is lost while positive control is activated. This promotes cell proliferation and thus wound healing. The rate at which this occurs is dependent on the factors mentioned above as well as a number of other proliferative factors such as growth factors that tend to be cell specific. 



Intracellular Controls


While mechanical trauma can influence cell proliferation, there are also different types of internal controls responsible for regulating this process. In particular, this has been shown to occur in cases where internal cell structures are affected.

In a case where DNA is damaged, then given processes depend on the stage of the cell cycle. A good example of this is when DNA is damaged during the G1 phase. 

Here, a protein known as p53, having identified mismatches in the DNA, activates another protein (p21) which in turn binds to the CDK–cyclin complex and consequently inhibits activities of Cyclin-Dependent Protein Kinase. As a result, target proteins of this enzyme are not phosphorylated which halts cell cycle. 

This is a good example of negative intracellular control given that it stops cell cycle and thus cell proliferation. However, following DNA repair, then the level of p53 protein drops allowing the activities of CDK to proceed and consequently allowing the cell cycle to proceed (positive intracellular control). 


Some of the other molecules and proteins that act as intracellular controls include:

 

  • Cytochrome c - involved in the activation of initiator caspases
  • Bcl-2 and Bcl-x - play a role in blocking the release of cytochrome c



Cell Cycle and Cell Proliferation


Essentially, cell proliferation (increase in numbers) occurs through cell division where a cell divides into two equal copies. Here, growth factors within a given environment employ various growth factor signaling pathways that not only influence/promote cell growth, but also activate these cells to enter the cell cycle. 

 

* Apart from being activated by growth factors, some cells also enter the cycle through the process of fertilization. 



Credit: Cell Proliferation Signaling Pathway HD Animation by 

McGraw-Hill Animations


Events of Cell Cycle


For resting cells (G0) or stem cells to enter the cell cycle process, they first have to be activated by growth factors. Once activated, the cells enter the first period of growth (G1).

At this stage, the cell starts preparing for DNA synthesis. Before the restriction point (R) which is the point towards the end of the first phase of cell growth, various factors can affect the process thus preventing the cell from entering the other phases of the cell cycle. 

Past this point, the cell is irreversibly committed to go through the remaining phases of the cycle. While growth factors play an important role in initiating the process, it's the internal cell signaling cycle that takes charge of the rest of the phases of the cell cycle. 

 

 

·       G1 phase (interphase) is the phase in which a variety of proteins are synthesized within a cell for DNA replication 

 

·       S phase is in which chromosomes are replicated to form two sister chromatids.

 

·       G2 phase - During this phase, different types of proteins required for mitosis (M phase) are synthesized. 

 

·       Stages of cell cycle (mitosis)

 

Mitosis (M phase) is a type of cell division that results in the production of two daughter cells.


The following are the main stages of mitosis: 

 

* Mitosis is initiated by cyclin B (CDK1)

 

·       Prophase  - During this stage of mitosis, the centrosomes that were initially paired start separating as the chromosomes start condensing in order to form sister chromatids (attached to each other by cohesion molecules of the centrosomes). It's also at this stage that the nuclear membrane starts to disintegrate.

 

·       Prometaphase - During prometaphase, the nuclear membrane that had started to disintegrate at the prophase stage disappear as spindle fibers start assembling. These fibers (microtubules) increase the space between the poles while also attaching the sister chromatids.

 

·       Metaphase - During metaphase, the microtubules start pulling the sister chromatids apart. However, they do not separate until all the chromatids are in place. 

 

·       Anaphase - Once all the sister chromatids are in place, a signal influenced by anaphase-promoting complex stimulates their separation.

 

·       Telophase - During this stage, the chromosomes completely separate and start moving to the opposite poles of the cell. Moreover, the cell starts to divide in a process known as cytokinesis. Here, contractile rings known as actomyosin cleave the cell into two ultimately producing two daughter cells. 

 


Cell Proliferation in Differentiation


As compared to differentiated cells, embryonic cells go through rapid proliferation. Once these cells differentiate, the rate of proliferation tends to decrease with some of the new cells only undergoing proliferation in order to replace those lost through injury, etc.

Here, however, it's worth noting that among adults, cells can be divided into three main groups with regards to proliferation. 


These include:

 

  • Cells that no longer divide following cell differentiation 

 

Following differentiation (among adults), some of the cells can no longer divide and proliferate. This includes such cells as those of the cardiac muscle (myocytes) and neurons. Having differentiated, these cells lose their ability to divide even in the event of injury where some cells are destroyed. They are therefore retained as they are during the lifetime of the patient and never replaced/replenished. 

 

  • Differentiated cells in adults that undergo cell division/proliferation

 

Among adults, the majority of cells enter the G0 phase and only undergo cell division when needed. These include such cells as epithelial cells of various internal organs and skin fibroblasts etc.

Following an injury, skin fibroblasts will undergo cell division in order to produce new cells to repair the damage (close the wound/cut). This is also the case with cells of the liver that tend to proliferate to replace lost tissue.

In mice, for instance, removing as much as two-thirds of the liver stimulates rapid proliferation that repairs the damage ultimately regenerating the entire liver. These cells, therefore, play a role in the repair of different types of tissues and organs. However, they only do so when necessary. 


 

As is the case with myocytes, blood cells and epithelial cells lining the digestive tract among others are incapable of dividing to form new cells. However, compared to myocytes, these cells can be replaced by a special group of cells known as stem cells.

These are less differentiated cells that undergo division to produce new daughter cells (stem cells that are less differentiated) or cells that can differentiate to replace dead or damaged cells.

For the most part, such cells as red blood cells have a short life span (a few days to several months) and have to be replaced. They are important cells to the body (e.g. red cells that transport oxygen to cells around the body) and therefore have to continue being replaced when the old ones die. However, because they are unable to divide, stem cells have to continue differentiating in order to replace lost cells.

It's also worth noting that once the stem cells differentiate, they also lose the ability to divide and will have to be replaced by newly differentiated cells from stem cells once they die. 

 

  • Cell proliferation and cancer

 

Under normal circumstances, cell proliferation is regulated by a number of controllers (positive and negative controllers) that activate and stop cell division. Therefore, in such cases, cells can only divide a given number of times (e.g. replace cells lost during an injury) before they die off or enter growth arrest (the G0 phase). 

Under certain conditions, abnormal proliferation may occur where cells continue proliferating unregulated. A good example of this is where abnormal cells divide uncontrollably to form a mass of cells (tumor) which may be cancerous. Here, the abnormal cells not only continue dividing more than they should, but also don't die. 

 

* The death and elimination of old and damaged cells is important in that it prevents the accumulation of cells that are incapable of carrying out their functions in a normal way.

 

Some of the factors that may cause DNA mutations (and thus abnormal cell proliferation) include:

 

  • Environmental artificial sources: e.g. organic chemicals and pesticides
  • Natural sources such as viruses and some toxins
  • High energy radiations such as gamma rays

 

Comparison of LDH activity in normal and cancerous cell metabolisms by Bcndoye [CC BY-SA (https://creativecommons.org/licenses/by-sa/3.0)]Comparison of LDH activity in normal and cancerous cell metabolisms by Bcndoye [CC BY-SA (https://creativecommons.org/licenses/by-sa/3.0)]

 

Currently, two mechanisms are used to describe how tumor cells are able to evade cell death.


These include:

 

  • The ability of tumor cells to maintain telomeres


A telomere refers to the sequence of nucleotides located at both ends of chromosomes. Telomeres play an important role in preventing the deterioration of chromosomes as well as preventing individual chromosomes from fusing to the neighboring chromosomes.

Under normal circumstances, telomeres become shorter following each cycle of cell division and are therefore described as cellular clocks in some books.

Once they are completely depleted, the cell can no longer divide as it enters the senescence state. While some of these cells are replaced (either by differentiating stem cells or mitotic cells) others are no longer replaced when they die (e.g. myocytes).

For a majority of malignant cells (about 90 percent of human tumors), however, high amounts of telomerase (an enzyme) continue adding DNA to the telomeres which prevents them from being depleted. As a result, the cells retain their ability to continue dividing over time. 

 

  • Alternative lengthening (ALT)

 

While a majority of human tumor cells rely on the high level of telomerase enzyme that prevents the depletion of telomeres, about 10 percent of the tumors have been shown to rely on a mechanism known as ALT (alternative lengthening of telomeres). In this mechanism, chromosome ends are replenished through homologous recombination.

For normal cells, this process involves the use of neighboring DNA (normal functioning DNA) as the template to repair another piece of damaged/broken DNA. For human tumor cells, however, the mechanism serves to replenish telomere DNA thus preventing their depletion (telomere depletion).

 

* According to a study that was funded by the National Institutes of Health and the Institute Merieux in 2011, a string of DNA containing high amounts of the base cytosine (C-tail) was found residing over the tip of telomeres of malignant cells and therefore suggested to contribute to the ALT mechanism.

 

Because of the ability of tumorous cells to continue dividing without dying, they continue to increase in numbers thus forming masses of cells. Unlike normal cells, these cells are abnormal and therefore do not perform normal cell functions.

This is particularly dangerous to the patient given that such cells may end up negatively affecting other normal cells and their functions.

For instance, in the case of leukemia, the bone marrow continues to produce abnormal white cells that don't function normally. As a result, the patient is likely to experience frequent infections that can be severe. 

 




  • Cytotoxicity assays

 

In medical sciences, cell-based assays are often used for the purposes of drug discovery, as well as measuring cell viability and proliferation. To determine the impact of different types of molecules (toxic agents) on cell viability and proliferation, cytotoxic assays are used. 

This is particularly important in drug screening and research on new types of drugs to test the impact of the molecules on cells (whether they present any cytotoxic effects etc). For the most part, the primary aim of such assays is to study cell viability following the tests as well as the general impact of the molecules on proliferation. 

 

* While these assays are important for drug screening and drug discovery, they are also used to determine the impact of different types of compounds in the environment. 

 

A good example of a cytotoxicity assay is the LDH Cytotoxicity WST Assay. As a colorimetric assay kit, this method works by measuring the activity of lactate dehydrogenase released from damaged cells. It's worth noting that lactate dehydrogenase  (LDH) is an enzyme found in all cells.

Following damage to the cell membrane, the enzyme is released into the surrounding environment where it can be measured. For this reason, it's often used as a biological marker in various cytotoxicity studies. 

 

Currently, a variety of cytotoxicity assays are available to measure the impact of various molecules on cell viability and cell proliferation. 


These methods are based on various cell functions including:

 

  • Enzyme activities: e.g. Quick Cell Proliferation Assay and LDH-Cytotoxicity Assay II
  • Cell adherence
  • Nucleotide update and DNA synthesis: e.g. BrdU Cell Proliferation Assay
  • ATP production, and Co-enzyme production: e.g. MTS Cell Proliferation Assay

 

Typically, different types of chemicals will have different cytotoxic effects on the cell. Whereas some of these mechanisms result in the destruction of the cell membrane, others may act by preventing the synthesis of important proteins within a cell. For this reason, different cytotoxicity assays are used to determine the specific impact of the chemical on cells. 


These assays are classified as follows:


 

  • Dye exclusion assays - e.g. erythrosine B assays

 

  • Fluorometric assays - e.g. amalarBlue assay

 

  • Colorimetric assay - e.g. XTT assay and WST-8 assay

 

  • Luminometric assays - e.g. ATP Assays



Four steps of Cell Signaling

What is Cellular Metabolism?

Return to Cell Biology main page

Return to Cell Differentiation

Return to Cell Division

Difference between Meiosis and Mitosis

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References

Arun Kumar. (2018). Why Study Cell Viability, Cell Proliferation and Cytotoxicity?

David Hughes, Huseyin Mehmet. (2004). Cell Proliferation and Apoptosis. 

Michael J. Berridge. (2012). Cell Cycle and Proliferation. Cell Signalling Biology.

Özlem Sultan Aslantürk. (2017). In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages.

 

Links 


https://www.ncbi.nlm.nih.gov/books/NBK9906/

https://www.nature.com/subjects/cell-proliferation



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