Expression of factors ruling proliferation, modified

Cell growth, division and proliferation are influenced by the action of external signals like peptide growth factors or hormones that bind and subsequently activate specific receptors. The activated receptors transmit the signal directly or indirectly, activating other substrates, to the cell nucleus.  Thus, the signals are converted into programmed responses of the cell, consisting of specific modification of gene expression (Vogt, 1993).

Proto-oncogenes have different functions, but they are all involved at different levels of signaling pathways that drive proliferation (Cline, 1987). Pro-growth proto-oncogenes might code for several proteins such as growth-factors, growth factor receptors transcription factors, signal transducers or cell cycle proteins (Cline, 1987; Robbins and Cotran, 2014). The expression of pro-growth proto-oncogenes are strictly controlled in normal cell; a disturbance of these controls may convert proto-oncogenes to oncogenes which are their constitutively activated form. Oncogenes encode oncoproteins that alter cell growth properties providing self-sufficiency in cell growth ultimately leading to oncogenic transformation (Torry & Cooper, 1991; Vogt, 1993). Proto-oncogenes that may promote cell growth when altered are the ones coding for tyrosine kinases and downstream signaling components such as RAS (Rat Sarcoma Virus), which is immediately recruited after the activation of the tyrosine kinase receptor, the downstream MAPK (mitogen-associated protein kinase) pathway and PI3K (phosphatidylinositol-3-kinase)/AKT (Protein Kinase B) pathway (Robbins and Cotran, 2014).

Growth factors: cells normally require stimulation by growth factors to proliferate. Soluble growth factors are produced by one cell type and act on a neighboring cell through a paracrine action to stimulate proliferation (Aaronson, 1991). Some cancer cells gain the ability to synthesize the same growth factor to which they are responsive, generating an autocrine loop (Shvartsman et al., 2002; Robbins and Cotran 2015). Growth factors promote entry of cells into the cell cycle, relieve blocks on cell cycle progression, prevents apoptosis, enhance the synthesis of all components are required to the formation of a new cell (Robbins and Cotran, 2014).

Transcription factors: all signal transduction pathways converge on the nucleus, where the expression of target genes involved in cell progression and mitotic cycle is activated; the consequence of altered mitogenic signaling pathways is deregulated and continuous stimulation of nuclear transcription factors that cause continue unopposed cell growth and proliferation (Baghwat & Vakoc, 2015). Hence, growth autonomy may happen because of mutations affecting the transcription factors that regulate the expression of pro-growth genes and cyclins (Robbins and Cotran, 2014).

Cyclins and Cyclin-Dependent Kinases: growth factors transduce signals that stimulate the correct progression of cells through the several phases of cell cycle which is the process by which cells replicate their DNA in preparation for cell division (Barnum & O’Connel, 2014; Wang, 2021). Progression of cells through the cell cycle is regulated by cyclin dependent kinases (CDK), which are activated by the binding to cyclins which called are called in this way due to the cyclic nature of their production and degradation. The CDK-cyclin complexes phosphorylate specific target proteins that lead cells forward through the cell cycle. There are also CDK inhibitors which role is silencing CDKs and exerting negative control over the cell cycle; the expression of these inhibitors is downregulated by several mitogenic signaling pathways, promoting the progression of the cell cycle (Barnum & O’Connel, 2014; Wang, 2021). There are two main cell cycle checkpoints one at G1 (growth phase)/S (DNA synthesis phase) transition and the other at the G2(cell growth phase)/M (mitotic phase) transition. Both cell cycle check points are tightly regulated by a balance of growth promoting and growth suppressing factors, as well as sensors of DNA damage; when activated, the DNA damage sensors cause the arrest of cell cycle progression and if the damage cannot be repaired, apoptosis is initiated. Defects in these checkpoints may lead to cancer development and progression (Shackelford et al., 1999; Robbins and Cotran, 2014).

Gene expression

Gene expression is directly determined by quantitating mRNA levels of the genes of interest through several established methods such as: (quantitative) real time reverse transcription polymerase chain reaction (q)RT-PCR), microarray expression profiling, whole transcriptome RNA sequencing. These two last methods allow the simultaneous quantitation of many different transcripts.

In situ hybridization localize specific RNA sequences in cells/tissues by means of complementary binding of a nucleotide probe to a specific sequence of RNA. The probes can be labeled with fluorescent- or antigen-labeled bases.

Luciferase Reporter Gene

The chemiluminescent reaction catalysed by luciferase is one of the most sensitive analytical tools for measuring gene expression identified. Because of the nature of the luciferase protein, its activity is directly measurable in in vitro translation, and in eukaryotic and prokaryotic transfection systems.

Protein expression

Protein expression can be measured in cells/tissue lysate as well as in situ. Most common methods are immunohistochemistry (in situ), western blot analysis and enzyme-linked immunosorbent assays (ELISA)

All living systems, physiology and pathology.

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