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Telomeres: Effect of Length of the Cell

Telomeres: being or immortal or getting cancer?
“Death takes place because a worn-out tissue cannot forever renew itself and because a capacity for increase by means of cell division is not everlasting but finite” said A.Weismann at Oxford in 1881 and without noticing that he made a brief description of the structure of what we now call as the telomere. The term is originating from Greek: telos means “end” and meros means “part” and currently the definition is more or less about telomeres being parts of a chromosome which are located at the end of them. Telomeres are repetitive tandem sequences that are protecting chromosome stability from degradation, “end fusion, and chromosome aberrant recombination.” (Hou et al. , 2012) They are also “postulated as a universal biological clock that shortens in parallel with aging in cells” (Oeseburg, 2009) Before elaborating on the shortening, lengthening and its outcomes I would like to give a brief historical background of these tiny but extremely important structures. Muller in 1938 first proposed these “end-parts” of chromosomes and then after 23 years Hayflick had the proof that cells were not immortal and had a limited number of times for undergoing cellular division. A Russian researcher called Olovnikov then made the connection between these two correct but thought to be uncorrelated notions. After this point, two scientists observed the sequence in 1978 in the end parts and lastly the identification of the sequence was made in the 80s by Robert Moyazis.
The problem known as the ‘end-replication problem’ which “describes the effect that linear chromosomes cannot replicate their terminal ends of the chromosome and consequently shorten at each mitotic cycle” (Oesburg, 2009) is actually the basic reason behind this mechanism telomeres being called as a biological clock. An evolutionary finding which was considered as a breakthrough in terms of telomere biology was found in 1985 by Carol Greider: the reverse transcriptase telomerase. This enzyme “in contrast to DNA-polymerase…is capable of elongating the telomeres” (Oeseburg, 2009) this means that a sequence can be created in the cell if necessary. If the end-replication problem is bypassed with the presence of this mentioned enzyme then theoretically the stability would be reached and furthermore no cell death would be necessary. This mechanism is actually how cancer cells are able to proliferate. Cancer is a disease caused by uncontrolled cell division in a part of the body. When the telomerase activity is more than what chromosome needs for stabilization the process turns into uncontrolled amount of cell divisions. These divisions occur so simultaneously that the replication efficiency drops gradually leading to damages or mismatches to occur. These mismatches results in mutations since their probability of happening are quite high. Mutations in addition to cells that have high telomerase activity therefore cause dysfunctional cells that are uncontrollably dividing. It comes to mind that if we are able to stop telomerase activity we could be able to eliminate the cancer disease from our lives. However, unfortunately inhibiting telomerase activity would again probably end with death. These evolutionary conserved sequences have some vital role in some of our specific cells.
The length should be stabilized in certain cells that need to divide as long as we are alive. For example there is this general knowledge that when you break an arm at a young age you recover well and soon however if you break your arm when you are old the recovering percent is low as well as the time it will take will be very long. This is somehow related with telomerase activity. The cells when you are young will have long sequences called to be the telomere. They will have large amount division chances so they will divide and help you recover. The older you get the shorter will be the telomeres and so no new cells will be there to heal you. The only types of cells that will divide until the end of one’s lifespan are stem cells, reproductive cells (until sometime but for a long period of your life) and lastly cancer cells. Even though we hope the cancer cells lose their telomerase’s at some point and diminish the deadly disease the other cells should be able to proliferate for us to be able to repair recombine etc.
Research I conducted basically demonstrates the recent founding on telomerase. First I will be comparing the length of telomeres and the effects they have over the cell. The discussion will basically depict the founding on short telomeres and disease it causes as well as long telomeres and the diseases it causes. Then I will elaborate on human telomerase RNA. Lastly I will mention the anti-telomerase cancer treatment and conclude my report.
According to Hou’s mini-review Surrogate tissue telomere length and cancer risk: Shorter or Longer? written in 2012 states that “the results have been inconsistent, showing positive, inverse or null associations between TL and cancer risks,” (Hou et al., 2012) however it is also stated that “differences in study design, biological sample collection and processing, specific cancer site, limited statistical power, variability in confounding factors, and differences in laboratory measurement of TL, may be contributing factors to inconsistent results in telomere association studies.” (Hou et al., 2012) Therefore it is very early to conclude on anything, more research has to be done to be sure of the causes and the effects to eliminate this risky factor out of our lives.
To begin with, shorter telomere is said to be associated with having a cancer risk more than usual. The studies are made amongst a lot of tissues such as breast, renal cell, bladder, lung etc. “Age-related diseases and premature ageing syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase.” (Blasco, 2005) and this finding is supported by another paper published in 2012: “When telomeres become critically shortened, cells undergo either replicative senescence or apoptosis. If such processes are bypassed, cells continue to proliferate through activation of telomerase, leading to genomic instability. The accumulated mutations, genetic lesions, and inactivated tumor suppressor checkpoints may ultimately result in cancer.” (L.Hou et al., 2012) says mini-review conducted in Cancer Letters, and it continues “Our investigation of TL in gastric cancer confirmed that factors increasing oxidative stress or inflammation, such as cigarette smoking, decreased fruit and vegetable intake, and chronic H.Pylori infection are associated with shorter TL in blood DNA.” (L.Hou et al., 2012) Therefore it is viable to say that environmental factors have a great but a partial effect in terms of shortening the telomere length and eventually causing cancer.
As for the long telomeres same review mentions the seven recent studies were submitting that cancer patients were exposed to long telomere length. Mostly breast cancer patients were diagnosed with having longer telomere than usual. It is stated that “prolonged estrogen exposure in breast cancer patients could be a possible reason for the longer TL observed in” (L.Hou et al., 2012) some of the studies on breast cancer. Furthermore it is also a fact that these patients have molecular components different than other people “patients with longer TL length had elevated levels of various telomerase-stimulating factors. (L.Hou et al., 2012) With all these data presented it is also plausible to say that longer telomerase may play a role in cancer as well.
When a closer look is taken at the molecular levels a study thought “it was surprising that the amount of hTR was high in cell strains that lacked telomerase activity, and the levels did not parallel the increase in telomerase activity, which accompanies immortalization.” (Avilion, 1996) hTR is the acronym for human telomerase RNA and it basically focuses on the interaction between the RNA responsible for making more telomere in the cell. What the researchers found out was this link. According to their findings the fact that hTR not being present in cell lines that did not lack telomerase activity meant that “RNA is not the limiting for telomerase activity, and that the RNA component is not a good predictor of the presence of enzyme activity.” (Avilion, 1996) The result of their experiments showed that telomerase activity was not present much in normal cells but would be detectable during the period in which the cell is in crisis. To demonstrate the statement above they used Northern Blotting and got the analytical data that supported their hypothesis. The conclusion was implying that telomerase activity was regulated in different levels rather than only one since none of the tumor samples they analyzed reflected the telomerase activity.
Lastly, the mentioned anti-telomerase cancer therapy has multiple targets but it is challenging due to the several different levels of regulations it has. What is unwanted is the drugs inhibiting the telomerase activity that is mandatory for a person’s daily life. Products, the drugs therefore, should have great specificity, low toxicity and few side effects. It is hoped that the outcomes would be far more positive compared to the initial state of the patient but as far as my research this is a field that is still refreshing itself and developing each and every second.

DNA Protein Cross-links in Human Saliva

Reproducibility and sensitivity issues of K-SDS assay-inter specific cohort studies
Suresh P.K[1]., Amrutha Kalyansunder*., Munmun Saha*
Abstract— A covalent bonding between a protein moiety and DNA molecule forms a DNA protein crosslink (DPC). Previous work on DPC’s has attempted to identify the specific proteins and create optimized techniques for their isolation to use in proteomic studies. Our experiment involves reproducing the K-SDS assay for DPC determination using exfoliated squamous epithelial cells (Squames) found in human saliva, and establishing DPC ratio within individuals of the same age group in a population to ascertain their possible role as a biomarker in conditions such as Oral sub mucous fibrosis (OSF).
Index Terms— DPC, K-SDS assay, Squamous epithelial cell, Biomarker, OSF.
Introduction DNA protein cross links have been known to occur along with single stranded breaks in cultured buccal epithelial cells and they have been investigated to study oral cancer growth in quid chewers[1]. Assays on DNA protein cross links have tried to establish the exact amount of covalently bound protein to DNA. By knowing the nature and amount of protein bound to the DNA it’s possible to determine the role of DNA-protein cross links.DNA of eukaryotes has the involvement of more proteins for controlling gene expression and protein synthesis pathways. So it is our assumption that certain protein moieties are bound to the DNA of Squamous epithelial cells which we have extracted.
DNA-protein crosslinks have been observed along with single stranded DNA breaks. Since DNA-protein crosslinks have been increasingly linked to heavy metal exposure[2,3] and Betel quid chewing it would be a good choice for a potential biomarker. However, this requires in depth research and understanding of the very nature of the crosslinks formed and it has not been validated in any former studies. We question the link between DPCs present in saliva and its association with pre cancerous conditions in the oral cavity.
Previous experiments on DNA-protein cross links have been partial to the use of human lymphocytes whereas salivary
epithelial cells have been the less common choice. It was our choice to proceed with salivary epithelial cells and check the sensitivity and reproducibility of a previously performed assay[4,5] to determine if similar results would arise with a different cell type and without external stimulation to establish a DNA standard for normal salivary cells.
[1] Corresponding address p.k.suresh@vit.ac.in
*Authors have equal contribution towards article
MATERIALS AND METHODS DNA Quantitation We determined the A260 absorbance for all the samples to ensure sufficient amounts of DNA was present in the cells. We assumed 1O.D = 50 ug/ml and calculated concentrations of DNA accordingly. We obtained values ranging from 1.0-2.3 ug/ml from which we inferred that DNA was relatively pure without interference from external sources and the protein contamination in samples was deemed insignificant.
Extraction of Squamous epithelial cells Samples were taken to perform a cohort study from 40 individuals (n=40) out of which 22 were male and 18 female of age group 22 3.Informed consent was obtained from all the subjects prior to experimentation. They did not consume food 2 hours prior to swishing.
25ml of 10X PBS buffer was swished 40 times on either side of the mouth for approximately 45 seconds and dispensed into two 15ml centrifuge tubes. They were centrifuged at 1500 rpm for 10 minutes. Supernatant was discarded taking care that the pellet is left undisturbed.
Cell lysis To the pellet 0.5ml of lysis cocktail* was added and incubated overnight at -70 0C. The samples are thawed at room temperature and dispensed through a 21 gauge needle 4 times to facilitate minimal foaming. 0.5ml of 100 mM Kcl,20 mM Tris-Hcl each were added to the samples and vortexed for five seconds at the maximum speed.
Samples were then heated in a boiling water bath for 10 minutes at 65 degrees. Its then carefully inverted 3 times and placed on crushed ice in an ice box for 10 minutes. They were centrifuged at 9000 rpm for 5 minutes at 4 0C.The supernatant was discarded and to the pellet 0.5ml of 100 mM Kcl, 20 mM Tris-Hcl was added. The washing, heating and centrifugation steps were repeated twice.
Protein digestion To the final pellet add 0.5ml of 100mM Kcl,20 mM Tris-Hcl(pH 7.5),10mM EDTA and 0.2 mg/ml Proteinase K . The samples were incubated at 50 0C for 2.5 hours with occasional shaking. The samples were then stored at -70 0C.
Protein Quantitation with Hoechst 32258 absorbance
To expose the proteins from DNA we divided each sample in to 2 aliquots of equal volume. To the second tube 50 micro liters of 4 mg/ml BSA was added and placed on ice for 30 minutes. Both aliquots were then centrifuged at 10,000 rpm for 10 minutes at 4 0C.The supernatants of the individual aliquots of both sets of tubes were taken in 2 ml microfuge tubes without disturbing the pellet.
Hoechst 32258 dye solution was prepared from a 1X stock solution by adding 30 micro liters to 100ml of 1X TNE buffer.1ml of the freshly prepared Hoechst solution is added to 1 ml of each aliquot in the microfuge tubes and mixed. The dye addition took place in a dark chamber and was left undisturbed for 10 minutes. For the readings, Jasco spectrofluorimeter FP-8200 was used. Excitation wavelength was set at 365 nm and emission wavelength at 440-600 nm in order to cover a wider range. The samples of both sets of aliquots were loaded into the cuvettes and readings were taken separately.
RESULTS DNA concentrations of the 22 male and 18 female controls were calculated as follows,
(*lysis cocktail=167 micro liters of 0.5% SDS, 20 mM Tris-Hcl and 1mM PMSF each)
Table 1
DNA Quantitation in male subjects
Males
Absorbance(nm)
Concentration(mg/dl)
1
0.0325
1.6
2
0.0365
1.8
3
0.0246
1.2
4
0.0300
1.5
5
0.0210
1.05
6
0.0421
2.1
7
0.0358
1.7
8
0.0320
1.6
9
0.0255
1.2
10
0.0251
1.2
11
0.0259
1.2
12
0.0420
2.1
13
0.0350
1.7
14
0.0311
1.5
15
0.0202
1.0
16
0.0360
1.8
17
0.0239
1.1
18
0.0254
1.2
19
0.0466
2.3
20
0.0369
1.8
21
0.0284
1.4
22
0.0278
1.3
Note: 1O.D=50 ug/ml of DNA
Table 2
DNA Quantitation in female subjects
Females
Absorbance(nm)
Concentration(mg/dl)
1
0.0395
1.9
2
0.0411
2.0
3
0.0299
1.4
4
0.0222
1.1
5
0.0412
2.0
6
0.0408
2.0
7
0.0231
1.1
8
0.0333
1.6
9
0.0283
1.4
10
0.0420
2.1
11
0.0399
1.9
12
0.0324
1.6
13
0.0257
1.2
14
0.0377
1.8
15
0.0261
1.3
16
0.0404
2.0
17
0.0313
1.5
18
0.0405
2.0
Note: 1O.D=50 ug/ml of DNA
Table 3
Results of Hoechst 32258 readings in Jasco FP-8200 spectrofluorimeter
Sample
Subset 1(nm)
Subset 2(nm)
DNA precipitation
1
177.324
279.846
0.6
2
196.515
294.323
0.6
3
237.548
416.358
0.5
4
238.586
491.147
0.4
5
1066.92
1910.73
0.5
6
1637.9
2474.85
0.6
7
1078.4
2101.06
0.5
8
1611.61
2004.21
0.8
9
704.147
2080.5
0.3
10
794.228
1420.88
0.5
11
970.45
1850.1
0.5
12
782.815
1625.88
0.4
13
778.678
1458.55
0.5
14
550.372
1095.21
0.5
15
422.632
1107.81
0.3
16
1065.22
1398.8
0.7
17
1051.04
1900.41
0.5
18
481.332
1092.33
0.4
19
396.221
717.338
0.5
20
860.24
1258.32
0.6
21
782.55
1425.33
0.5
22
187.9484
990.218
0.1
23
111.501
860.24
0.1
24
431.561
1229.77
0.3
25
259.58
550.36
0.4
26
280.451
444.316
0.6
27
251.602
430.578
0.5
28
699.58
1422.33
0.4
29
500.537
1454.34
0.3
30
336.233
954.29
0.3
31
577.619
1278.02
0.4
32
867.856
1625.58
0.5
33
439.613
1431.53
0.3
34
1158.4
3002.03
0.3
35
1249.9
2954.58
0.4
36
1120.75
3520.33
0.3
37
809.063
1450.35
0.5
38
788.253
1268.03
0.6
39
772.399
1239.19
0.6
40
685.363
982.662
0.6
Note: Subset 1 refers to the samples without
BSA addition whereas subset 2 has the
presence of 4mg/ml BSA.

Fig. 1.The scatter plot represents DNA concentrations of Male subjects measured with absorbance at A260.R2 value is represented in the graph

Fig.2.The scatter plot represents DNA concentrations of Female subjects measured with A260 absorbance.R2 value is denoted in the graph

Fig3.Subset 1 and 2 are represented in X-axis with their respective absorbances in Y-axis. Subset 2 shows a marked increase in DNA concentration and contains 4mg/ml BSA addition.
Statistical analysis was done for both male and female samples and the mean and standard deviation were calculated as [1.38 0.32% (S.D.)] for males and [1.6 0.32% (S.D.)] for females respectively. A T-test was performed and the calculated value was 2.55.
Normalization of the 40 control samples was calculated as 0.4 0.5. It was done to check the correctness of the experiment and to reduce the redundancy of sample measurements.
DISCUSSION DPCs have been established as an indicator of DNA damage in body cells. Its concentration is known to be particularly high in cases involving formaldehyde interaction, heavy metal exposure, betel quid chewing and cells showing high levels of oxidative stress. Most of the current work involves studying specific populations showing DNA-protein cross-links or inducing them through various means to investigate their nature. [6]
Our experiment showed similar DNA concentrations in male and female subjects. We concluded that there was not much statistical significance in the amount of DNA extracted from normal individuals of same age group. [7] A two-fold increase in DNA content was observed in samples which were treated with BSA as opposed to samples which had no BSA. This is further proof to existing studies of DNA-BSA interactions.[8]BSA is a complex protein that has specific binding sites for a variety of metallic complexes[9] and stimulants.BSA is a model protein which is very similar to Human serum albumin(HSA) present in the human body. We can make an assumption that there has been precipitation of DNA due to the external addition of BSA. Further studies have to be made with clinical samples to see whether similar precipitation of DNA occurs with BSA treatment.
If the assays involving the quantification of DNA-protein interactions are perfected they could successfully be used as biomarkers for cancer research and chemical exposure specially to denote pre cancerous conditions. Our study aims to standardise the K-SDS assay in squamous epithelial cells taken from the oral cavity of normal individuals. We will be further comparing these results against areca nut chewers. We will be investigating whether or not chewing induces the formation of DPCs in such individuals. This will give insight for the use of DPC as a biomarker for cancerous conditions such as OSF and areca nut as an oral cancer stimulant.
We have encompassed samples representing both male and female individuals so that there is no bias in sampling and reproducibility of the assay is achieved. Sample collection is simple and storage is possible up to a limited period of time. The method is rapid and relatively inexpensive.
Acknowledgment We would like to express our gratitude towards Dr. Anbalagan and Dr. Bhaskar Mohan Murari, Center for biomedical research, VIT University, Vellore for proving us with the essential chemicals for our work. We would also like to extend our regards to the individuals who contributed their samples towards our study. We would like to humbly acknowledge Professor P K Suresh of VIT University for encouraging and guiding us through every step of this study.
References [1] Kristina Sundqvist, Yun Liu, Jagadeesan Nair(1989) Cytotoxic and genotoxic effects of Areca-Nut related compounds in cultured human buccal epithelial cells Cancer Res;49:5294-5298.
[2] Pativrno S.R. and M.Costa (1985) DNA-protein cross-links induced by nickel compounds in intact cultured mammalian cells.Chem.Biol.Interact. 55, 75-91.
[3] S.Barker, M Weinfield and D.Murray (2005) DNA-Protein crosslinks: Their induction, repair, and biological consequences.Mut.Res. 589,111-135.
[4] A. Zhitkovich and M Costa (1992) A simple and sensitive assay to detect DNA protein crosslink in intact cells and in vivo. Carcinogenesis. 13, 1485-1489.
[5] M.Costa, A. Zhitkovich, M. gargas, D Paustenbach, B.Fentley, J Kuuykendall et. al (1996) Interlaboratory validation of a new assay for DNA-protein cross-links, Mut. Res. 369, 13-21.
[6] S.Barker, D.Murray, M.Weinfeld et.al (2005), A method for the isolation of covalent DNA-protein cross-links suitable for proteomics analysis, Anal.Biochem 344,204-215.
[7] M. Costa, A.Zhitkvoich and P toniolo (1993) DNA-Protein crosslinks in welders: Molecular implications.Can.Res.53, 460-463.
[8] Rajesh Chakraborty, Sriparna Chatterjee, Sandipan Sarkar3, Pabitra Chattopadhyay (2012) Study of Photoinduced Interaction between Calf Thymus-DNA and Bovine Serum Albumin Protein with H2Ti3O7 Nanotubes, J.Biomats .Nbtech, 2012, 3, 462-468.
[9] Laura Luzuriaga, María Fernanda Cerdá (2012) Analysis of the interaction between [Ru(phenanthroline)3] 2 and bovine serum albumin, Adv.Bio.Chem., 2012, 2, 262-267.

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