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Telomerase Activity in HeLa

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Mark Brown

on 14 November 2013

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Transcript of Telomerase Activity in HeLa

Telomerase Activity in HeLa
Cervical Carcinoma
Cell Line Proliferation

Goal
The goal of this research paper was to determine the telomerase activity in the dividing and non-dividing/senescence cells in HeLa cell cultures and calculate the reasons behind these differences, as-well as gathering useful information for future research.
Abstract-Somatic Cells
Somatic cells divide a finite number of times until they undergo senescence.
This occurs when the telomeres become too short and result in fusion of chromosomes, or they come under some type of biological stress.
The critical telomere length is 12.8 repeats (of six base pairs) long.
The vertebrate telomere has the sequence TTAGGG.
HeLa Cells
Unlike Somatic Cells, HeLa cells contain telomerase.

Telomerase is an enzyme that prevents the degradation of telomeres. This makes the cells immortal. Approximately 85% of cancer cells have active telomerase.

There are some HeLa cells that do not have telomerase. This is shown by the radioactive nucleoside 3H-Thymidine.
Materials, Methods and Results
SA-beta-Gal and 3H-thymidine labeling index- most normal human cell cultures gradually cease dividing. Senescent cells can't incorporate 3H-thymidine and SA-beta-Gal is an enzyme only present in senescent cells. Four morphologies produced.
Abstract
3-H Thymidine
Is a radioactive nucleoside, 3H-thymidine, that becomes incorporated into new strands of chromosomal DNA during mitotic cell division.

Testing the presence of radioactivity shows if the cell has divided or not.
Materials, Methods
and Results
Senescence versus quiescent state. Division upon sub-cultivation, lack of SA-beta-Gal.
In order to separate the non-dividing/Senescent researchers used fast-dividing DiI staining and high-speed cell sorting techniques.
Abstract: SA-beta-Galactosidase
Most of the time it is known as SA-beta-Gal, but it stands for, "Senescent Associated Beta Galactosidase".
This enzyme is only present in cells that are not dividing and are in a Senescent stage.
Testing of the non-growing cells showed a low SA-beta-Gal and high labeling index when it was sub-cultured again.
Webster's Definition
Senescence: "The state or process of being or becoming old."





Abstract-Telomerase
Telomerase is an enzyme made of protein and RNA subunits that elongates chromosomes by adding TTAGGG sequences to the end of existing chromosomes.

They are present in most HeLa cells and are the cause of them being "Immortal".
Experimentation
The third part of the experiment determined that a majority of the non-dividing cells had actually retained their ability to divide.
The results showed that about 28% of the L fraction(fast dividing) had the [3H]dT(-)/SA-beta-Gal(-), and the R fraction(non-dividing) had about 66% [3H]dT(+)/SA-beta-Gal(-)
Results
Most cells showed the typical [3H]dT(+)/SA-beta-Gal(-) phenotype. 90.11% (Dividing/not Senescent)
Second largest group was [3H)dT(-)/SA-beta-Gal(-). 9.47% (Not dividing/Not Senescent) Delayed S phase
The third group: [3H]dT(+)/SA-beta-Gal(+). 0.15%(Dividing/Senescent) Stress or Spontaneous Senescent after last division
The fourth group: [3H]dT(-)/SA-beta-Gal(+). 0.28%(Not dividing/Senescent) Normally expected
Additional Testing
(R fraction)
To further study the effects of senescence on the non-dividing strain of cells, further culturing was done and analyzed cells showed that virtually all cells continue to divide upon sub-cultivation. This confirms that [3H]dT(-)/SA-beta-Gal(-) phenotype cells are really just delayed entrance to the S phase cells.
Materials, Methods and Results
Separation of cells based on their ability to divide quickly or not divide was completed next using DiI labeling.

Fast dividing cells are labeled
(L fraction)
Non-dividing cells are labeled
(R fraction)

Further SA-beta-Gal testing took place. Sub-cultured to determine Senescent versus temporary senescent.
Discussion
Ultimately, the cells that aren't dividing may be temporarily not dividing based on stress or other inhibitors, but they definitely aren't all in senescence.
Telomerase activity is higher in cells that aren't dividing.
We still do not know why telomerase levels fluctuate.
Cancer research should be careful.
Abstract-DiI Labeling
DiI labeling is a detailed process in which a dye is incorporated into the cell's inner and outer membranes. This dye is fluorescent and with each cell division, the resulting daughter cell has 50% of the dye. This allows for separation of quickly dividing cells and non-dividing cells.

This labeling resulted in cells that divided vigorously and some that didn't divide.
Cells that didn't divide rigorously were found to contain much more telomerase activity.
Experiment
Testing the Telomerase activity in the fractions was the next step and the results were somewhat surprising.
The results showed that telomerase activity was about 4 times higher in the R fraction compared to L fraction. This gave reason to believe that high telomerase activity could lead to cell arrest or spontaneous senescence.
Discussion
Determining the percentage of non- proliferation- 3H-thymidine labeling. 90% divided, 10% didn't.
Used SA-beta-Gal to determine if it was Sesescent. 0.28% were.
To specifically analyze non-dividing cells- DiI labeling and high speed sorting. 6 days cultured. 0.15% of cells positive for radioactive and SA-beta-Gal.
Comparative, the majority of R fraction was in temporary growth stage. 96.7%
Growth arrest is temporary, and can last at least 6 days after which cells can regain dividing capacity.
Quick Overview
HeLa cell samples were tested for division via 3H-Thymidine.
The different types of dividing and non-dividing cells were analyzed using DiI labeling and assorted.
Telomerase activity showed that non-dividing cells had a high proportion of telomerase.
Further testing showed that non-growing fractions of cells had a high index label and low SA-beta-Gal upon second culturing.
Telomerase and Cancer
Works Cited and Authors
This research project was performed and written by Milena Ivankovic, Andrea Cukusic, Ivana Gotic, Nikolina Skrobot, Mario Matijasic, Denis Polancec, and Ivica Rubelj.

Works Cited

Ivanković, Milena, et al. "Telomerase Activity In Hela Cervical Carcinoma Cell Line Proliferation." Biogerontology 8.2 (2007): 163-172. MEDLINE with Full Text. Web. 14 Nov. 2013.
Materials, Methods, and Results
These cells that did not show the SA-Beta-Gal in the subculture prove that the "non-dividing" cells actually retained their ability to divide when sub cultured again after separation. This implies that most of the non-dividing cells in HeLa culture actually represent cells with delayed entrance into the S phase. Approximately 0.3% of cell are true senescence cells.
Cancer and Telomerase
The concern with Telomerase prohibiting cancer developments is that there is a delay of up to 20 divisions before the Telomerase inhibiting stops the proliferation and subpopulations can avoid influence of telomerase inhibitors and under a change of conditions can continue to replicate.
Summary
Telomerase activity in HeLa cervical carcinoma cell line proliferation varies depending on the type of fraction it is and whether or not it is sescent or not. We still do not know why telomerase activity fluctuates but it is interesting to know that non-dividing cells show more telomerase activity than the dividing ones.
Telomerase Activity Testing
In this particular research project the researcers used the TRAPeze Telomerase Detection Kit. This kit provies a mixture that is added to the lysed cells which allows for the elongation of TS primer. The TS primers allow for a formula to be used to quantify the number of Telomerase product.
Introduction
Spontaneous appearance of senescent cells documented in some tumor populations.
Fractions of these cells are divided by DiI and cell sorting.
Isolated fractions are sub-cultured and analyzed for growth and telomerase.
Results suggest that level of telomerase plays in imporant role in spontaneous appearance of senescent cells in HeLa line.
Estimated age... #error
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