Animal Cell Culture Lecture 6

Animal Cell and Tissue Culture Lecture 6

• SIO2004 is the course code for Animal Cell and Tissue Culture, taught by Dr. Nuradilla Mohamad Fauzi at Universiti Malaya. Lecture 6 is part of the Biotechnology Program.

Can Normal Cells Divide Forever?

• Most normal cells have a finite lifespan in culture.
• They will eventually stop dividing.

Replicative Cell Senescence

• Multicellular organisms replace worn-out cells through cell division.
• Proliferation slows in many cells until division eventually halts, entering a non-dividing state called senescence.
• In humans, this typically occurs after an average of 52 divisions, known as the Hayflick limit.
• Primary cells in culture have a limited lifespan.
• Cells stop dividing due to telomere shortening. Telomeres are protective DNA sequences at chromosome ends.

Telomeres

• Telomeres are repetitive nucleotide sequences at the ends of chromosomes.
• They protect chromosomes from deterioration or fusion with neighbouring chromosomes.
• They act as disposable buffers, preventing the truncation of the genes when replicating.
• In vertebrates, the telomere sequence is TTAGGG.

Telomeres and Cellular Aging

• Each cell division results in some telomere loss (typically 25-200 base pairs).
• In humans, average telomere length decreases from around 11 kilobases at birth to less than 4 kilobases in old age.
• DNA replication enzymes cannot replicate the entire chromosome, causing shortening in each duplication cycle.
• Short telomeres result in a "critical length" – the cell can no longer replicate, entering a senescence or apoptosis state.

Telomerase

• Telomerase is a reverse transcriptase enzyme that adds telomere repeats to DNA strands.
• It prevents chromosomal end degradation after multiple replication rounds.
• Active in fetal tissues, adult germ cells, and stem cells, but usually deficient in most post-natal somatic cells

Cells that Express Telomerase

• Fetal tissues, adult germ cells, stem cells, and cancer/transformed cells express telomerase.
• Telomerase expression allows senescent cells, that otherwise become post-mitotic and undergo apoptosis, to exceed the Hayflick limit and become potentially immortal (transformation).

Activation of Telomere Extension

• Telomere shortening, senescence, and apoptosis occur when telomeres are too short.
• Inactivation of DNA checkpoints, accumulation of mutations, and cancerous transformation lead to cellular immortality.

Transformation

• Transformation is a spontaneous or induced permanent phenotypic change in cultured cells.
• This is due to a heritable change in DNA and gene expression, contributing to genetic instability.
• Immortalization, loss of contact inhibition, aberrant growth control, density limitation, malignancy are characteristics in cell lines.
• Injected into animals with non-functional immune systems, transformed cell lines can cause tumors.
• Immortalization versus transformation, while related, are not identical.

Transformation and Cancer

• Transformed cell lines are in vitro equivalents of cancerous cells.
• Cancer occurs when a somatic cell, normally unable to divide, undergoes mutations.
• This deregulation leads to uncontrolled proliferation. Immortalized cell lines exhibit similar mutations, enabling a cell type not normally capable of dividing to proliferate in vitro.

Transformation Methods

• Natural/spontaneous (mutations in tumor/cancer cells, random spontaneous mutations in culture)
• Artificial expression of key proteins required for immortality (e.g., oncogenes, telomerase)
• Artificial inactivation of tumor suppressor genes (e.g., p53, Rb)
• Chemical mutagens (treatment of primary or non-transformed cell lines with chemical carcinogens, DNA methylation inhibitors)
• Introduction of viral genes (inserting viral genes that partially deregulate the cell cycle, such as SV40 T antigen, HPV-16 E6/7 gene)
• Tumor suppressor genes (ON in normal cells, OFF in cancer cells)
• Oncogenes (OFF in normal cells, ON in cancer cells; examples include telomerase)

Tumor Suppressor oncogenes

• Tumor suppressor - ON in normal cells, OFF in cancer cells
• Oncogene - OFF in normal cells, ON in cancer cells

SV40 T Antigen example

• The SV40 LT gene is used to induce immortalization.
• Its product, T antigen, binds Rb and p53, restricting DNA surveillance.
• This allows an increased chance of genomic instability, and subsequent mutations leading to immortalization (e.g. the upregulation of telomerase or the downregulation of a telomerase inhibitor).
• This process allows an extended proliferative life span.

Examples of Immortal/Cancer Cell Lines

• HeLa cells (cervical cancer)
• 3T3 cells (mouse fibroblast)
• A549 cells (lung cancer)
• Jurkat cells (leukemia)
• Vero cells (monkey kidney)
• F11 cells (neurons from rats)
• HEK 293 cells (human fetal cells)

Advantages of Cancer/Immortalized Cell Lines

• Cells can be grown indefinitely in culture.
• Inexhaustible supply of cells for research projects.
• No need to re-establish fresh primary cultures.
• Easy culture (no complex medium or additives).
• In vitro model for cancer.

Limitations of Cancer/Immortalized Cell Lines

• Genome instability (abnormal chromosomes change genetically over multiple passages). This leads to accumulation of mutations & variability.
• Poor model of normal tissues (even though often originating from a normal tissue type, mutations can alter cell biology and analysis needed)

Definition of Stem Cells

• Stem cells are capable of self-renewal (multiple division cycles maintaining undifferentiated state) and differentiation (capacity to mature into specialized cell types).

Importance of stem cells in vivo

• Stem cells are the body's raw material for generating other cells with specialized functions.
• They are essential for cell renewal (producing new cells to replace dead ones, normal wear & tear).
• They play a crucial role in the body's development & repairing damaged or diseased tissue

Importance of stem cells in research

• Stem cells provide valuable tools for cellular studies.
• They are used to increase understanding of disease processes.
• Stem cell therapy's regenerative medicine and transplantation are used to repair damaged tissue.

Cancer Stem Cells

• The "cancer stem cell" (CSC) hypothesis proposes that cancers are perpetuated by a small population of cancer stem cell-like cells.
• Researchers now believe cancer stem cells may trigger cancer growth and reproduction.
• These cells may survive treatment, which could lead to tumor relapse.

Evidence for Cancer Stem Cells

• No definitive proof for either theory, but there is increasing evidence suggesting the cancer stem cell theory holds true in some cases.
• Leukemia research showed only a subset of leukemia cells can cause further leukemia in a healthy body.
• Various cell types, like breast, brain, skin, prostate, and colonic cancers, have been found to contain stem-like cell characteristics.

Potential, Cell, Source Summary

• Totipotent: zygote, can differentiate into any tissue, from the zygote
• Pluripotent: embryonic stem cells, can differentiate into all three germ layers
• Multiopotent: Adult stem cells, differentiate into several tissue types
• Oligopotent: differentiate into a few tissue types
• Unipotent: differentiate into one cell type (e.g. fibroblasts)

This quiz covers Lecture 6 of the Animal Cell and Tissue Culture course, focusing on normal cell division, replicative cell senescence, and the role of telomeres. Explore the mechanisms behind cell lifespan and the Hayflick limit, crucial for understanding biotechnology applications in cellular biology.