Original Research Paper

Published: 04 March 2024

Arzu Erol

Genome Instability & Disease  Volume 5, (2024)

Abstract

The cause of cancer is disorders and unstable genome. Some factors affect proper functioning of the DNA. These situations lead to cell death, cancer, inborn disorders, and overall functional decline contributing to aging. The integrity of the genome of all living organisms is constantly threatened by exogenous and endogenous DNA-damaging agents. An agent might interact directly with DNA and regulate the expression or directly by influencing DNA repair responses and so improve genomic stability. The development of widely applicable methods to monitor genomic instability gains importance nowadays. RAPD-PCR assay is a molecular method able to detect comparative DNA changes. This work is to show the applicability of the method to evaluate the ultimate changes caused in various cell-culture-derived DNA that might be a model for carcinogenesis, genomic instability and routine cell-culture work.

Introduction

Preserving genomic integrity is obviously important, as manifested by our genome’s investment of some 250 genes for purposes of DNA damage repair (Akgün et al., 2022), more than 230 genes for high-fidelity DNA replication, and perhaps more than 500 for chromosome segregation, cell cycle checkpoints, telomeres, centromeres, and damage sensing (Burhansand et al., 2007).

Endogenous and exogenous DNA damaging agents are constantly challenging the integrity of the genome. If DNA damage is repaired incorrectly, it can lead to genome instability, which is associated with tumor genesis in human (Katerji & Duerksen-Hughes, 2021). Eukaryotic organisms have evolved several repair and surveillance mechanisms that remove DNA damage and coordinate cell cycle progression (Hoboken et al., 2001).

The cell cycle continues by a characterized grouping of occasions where late occasions rely on fruition of early occasions (Liu et al., 2024). The point of the reliance of occasions is to disperse finish and exact copies of the genome to daughter cells (Hoboken et al., 2001). To screen this reliance, cells are furnished with the checkpoints that are set at different phases of the cell cycle. At the point when cells have DNA harms that must be repaired, cells enact DNA harm checkpoint that captures cell cycle (Hong et al., 2005).

In spite of the fact that it may appear that immediate inversion of harm would be the least complex approach to amend the harm, by and large the invert response is impractical due to the thermodynamic or active reasons. In a couple cases, the response is reversible, and in some of these cases, systems have been produced to exploit that reversibility. Albeit all, cells have a substantial number of various sorts of repair framework, each moderately particular for a specific kind of DNA harm, those repair frameworks can be gathered into four noteworthy classes: Mismatch Repair, Base Excision Repair, Nucleotide Excision Repair, and Double-strand Break Repair (Sancar et al., 2004).

There are two important types of error in DNA. DNA damages are physical abnormalities in the DNA, such as single- and double-strand breaks, 8-hydroxydeoxyguanosine residues and polycyclic aromatic hydrocarbon adducts (Senavirathne et al., 2023).

Proteins can perceive DNA harms, and in this manner, they can be effectively repaired if repetitive data, for example, the undamaged grouping in the integral DNA strand or in a homologous chromosome, are accessible for replicating (Araten et al., 2005). In the event that a cell holds DNA harm, interpretation of a quality can be forestalled, and in this way, interpretation into a protein will likewise be blocked. Replication may likewise be blocked or potentially the cell may kick the bucket. Rather than DNA harm, a transformation is an adjustment in the base grouping of the DNA (Altonen et al., 1994).

RAPD remains for Random Amplification of Polymorphic DNA. It is a kind of PCR response, yet the portions of DNA that are enhanced are arbitrary. The researcher performing RAPD (Castano & Becerril, 2004) makes a few self-assertive, short groundworks (8–12 nucleotides), then continues with the PCR utilizing a vast format of genomic DNA, trusting that parts will increase. By settling the subsequent examples, a semi-interesting profile can be gathered from a RAPD response (Atienzar & Jha, 2006).

No learning of the DNA succession for the focused on quality is required, as the groundworks will tie some place in the arrangement, however, it is not sure precisely where. This makes the strategy well known for looking at the DNA of natural frameworks that have not had the consideration of established researchers, or in a framework in which moderately few DNA arrangements are analyzed. Because of the way that it depends on an expansive, in place DNA layout arrangement, it has a few restrictions in the utilization of debased DNA tests. Its settling force is much lower than focused, species particular DNA correlation techniques, for example, short pair rehashes. As of late, RAPD is utilized to portray, and follow, the phylogeny of assorted plant and creature species (Atienzar et al., 2000).

The study we put forward here is to our knowledge a novel approach in assessment of various DNA effects: either DNA damages or mutations caused by endogenous or exogenous exposures in routine cell-culture work (Keshava et al., 1999; Keshava et al., 2001; Lery et al., 2003; Lee et al., 2007) The various cell types either cell-lines or primary cells are for the sake of modeling various aspects of a cell’s life-time exposures and events and in this context to discuss the events such as genomic instability, DNA damage, mutation, DNA repair, mutagenesis, carcinogenesis, aging and related disorders. Below is a brief information before discussion of the results obtained that is to clarify the events underlying the work starting with the importance of genomic stability, how to maintain it, what happens when it is lost, what are the causes of genomic instability, what it may lead to in a cell’s life-time exposures.

Materials and methods

Cell types

Frozen cell lines (HEK293, HUVEC and HeLa) were purchased from American Type Culture Collection (ATCC). Collected cell-lines were stored in liquid nitrogen. All cell line culture samples were collected by American Type Culture Collection (ATCC) laboratory. All the information about cell-lines are provided in Table 1 from ATTC’s website.

Cell culture chemicals and consumables

Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640 Medium, essential amino acids, penicillin/streptomycin, Trypsin/EDTA, and fetal bovine serum (FBS and for mesenchymal cells: hMSC qualified FBS) were purchased from Biochrom, Berlin, Germany. Cell culture flasks: 15 and 50 ml polycarbonate centrifuge tubes with lids and cryotubes were from Grainer Bio-One Corp., Germany. Cell lines were cultured at 37 °C under 5% CO2 in culture medium unless otherwise specified (Caputo, 1996; Mather & Barnes, 1998).

Thawing cell lines

Previously frozen cells or cell line in liquid nitrogen was taken out from the tank, immediately soaked into pre-warmed water bath and incubated in water bath at 37 °C until totally thawed. Thawed cell gently mixed by pipetting and transferred to 15 ml tube that was containing 10 ml growth medium. Then, cells were centrifuged at 1500 rpm for 10 min and supernatant carefully aspirated. Lastly, cell pellet was resuspended in 1 ml medium and cell number was counted. After this, cells were seeded in 25 cm culture flask with 8 ml 10% FBS growth medium and were transferred to 25 cm sterile culture flask. Culture flask size was determined according to total cell number. Culture flask was incubated at CO2 incubator under humidified microaerofilic condition with 5% CO2. Following day, cells were observed under invert microscope and growth medium was refreshed.

Sub-culture of cell lines

Cultured cells were observed under microscope and if the confluency was about 90% and if there was no contamination or other abnormal formation (Chen, 1977), cells were determined to be sub-cultured. Culture medium was aspirated and cells were washed for once with pre-warmed PBS at 37 °C. After washing cells, they were detached by treatment with Trypsin/EDTA solution for 1–3 min until all cells were detached. 3 ml FBS for 25 cm sterile culture flask was added to detached cells to stop the Trypsin /EDTA activity. Then, cells were splitted or diluted depending on the purpose, transferred to new culture flasks, and incubated in 5% CO2 incubator at 37 °C.

Cryopreservation of cell lines

Medium of the cells that are 80–90% confluent was aspirated, the cells were washed with 5 ml pre-warmed PBS in 25 cm culture flask for once, treated with 1 ml Trypsin/EDTA (4 ml/25 cm), and incubated at 370 °C for 1–3 min to be detached. To inactivate Trypsin/EDTA, 3 volumes of 1 ml FBS were added. FBS plus Trypsin/EDTA mixture was transferred to 15 ml falcon tube and then was centrifuged at 1500 RPM for 5 min. After centrifugation, the cell pellet was resuspended with FBS and counted with hemocytometer. About 1.5–2 × 10 cells were added to each tube with 10% DMSO on ice and was left at − 20 °C for a while to cool down. Cryovials were transferred to − 20 °C immediately and left for 2 h, then transferred to − 80 °C and left overnight. At last, cells were transferred into liquid nitrogen.

DNA isolation

Macherey Nagel’s MN Nucleospin Tissue Kit was used and the components are as follows. Lysis Buffer, Buffer B1, Buffer B2, Wash Buffer B5, Wash Buffer BW, Elution Buffer BE, Proteinase K (lyophilized), Proteinase Buffer PB. DNA extraction was performed according to MN-Nucleospin tissue kit protocol step by step as describe below: 1.0 × 10⁶ cells were resuspended in a final volume of 200 μl Buffer T1. 25 μl Proteinase K solution and 200 μl Buffer B3 were added. Samples were incubated at 70 °C for 10–15 min. To adjust binding condition, 210 μl ethanol (96–100%) to the sample was added and vortexed vigorously. For each sample, one NucleoSpin^® Tissue Column was placed into a collection tube. The sample was loaded onto the column, was centrifuged for 1 min at 11,000g. Flow-through was discarded and the column was placed back into the collection tube. 500 μl Buffer BW was added onto the column, centrifuged for 1 min at 11,000g. Flow-through was discarded and the column was placed back into the collection tube. 600 μl Buffer B5 was added onto the column, centrifuged for 1 min at 11,000g. Flow-through was discarded and the column was placed back into the collection tube. To dry and remove the residual ethanol from membrane empty column was centrifuged for 1 min at 11,000g. NucleoSpin^® Tissue Column was placed into a 1.5 ml microcentrifuge tube and 100 μl prewarmed Elution Buffer BE (70 °C) was added onto the column, incubated at room temperature for 1 min, centrifuged 1 min at 11,000g. Highly pure DNA is extracted at the end of procedure and eluted DNA concentration and purity is calculated using Qubit Fluorometer.

DNA quantification

DNA quantification was done using Qubit fluorometer and its quantification kit components. Quant-it dsDNA BR Assay Kits Components are as follows: Working solution, Standard #1 Standard #2 and assay range is 2–1000 ng with sample starting concentration range: 100 pg/μl–1 μg/μl.

To determine concentration of the original samples, the instrument read values were multiplied by the dilution factor. Alternatively, calculate sample concentration can be chosen to have the Qubit fluorometer perform this multiplication.

Polymerase chain reaction PCR

RAPD is different from conventional PCR, as it needs one primer for amplification (Jones & Kortenkamp, 2000). The size of primer is normally short (10 nucleotides), and therefore, less specific, show in Table 2. The primers can be designed without the experimenter having any genetic information for the organism being tested. More than 2000 different RAPD primers can be available commercially.

Table 2 Base sequences of 10-mer primers

The conditions of RAPD-PCR were optimized with some modifications stated in Fig. 2 as RAMD-PCR (Random Amplified Mosaic DNA-PCR). RAPD-PCR reactions were performed in reaction mixture of 25 μl containing the components in Table 3.

Table 3 RAPD-PCR solutions and their initial and final concentrations with final volumes calculated accordingly

The RAPD and RAMD protocols consisted of an initial denaturing step of 5 min at 94 °C, followed by 45 cycles at 94 °C for 30 s (denaturation), 37 °C for 60 s (annealing) and 72 °C for 60 s (extension), with an additional extension period 10 min at 72 °C.

Agarose gel electrophoresis

PCR products were resolved on 2% agarose gels. Gel is prepared adding 1.6 g of powdered agarose gel into 80 ml of 0.5 × TBE buffer solution and it is boiled until the agarose is completely dissolved in the buffer solution in Table 4. 4 μl of Ethidium Bromide was added when the boiled solutions began to cool down and reach approximately 55 °C. Solution is mixed homogenously by making hand-shaking. It is directly poured into horizontal agarose gel platform and the combs having either 8 or 22 wells are placed one side of the gel. Gels are let to solidify for at least 10 min, to confirm full polymerization 45 min. Loading and visualization of the gels were done accordingly: 10 μl of PCR product was mixed with 2 μl bromophenol blue as loading dye/buffer. 12 μl PCR mixes were then loaded in each slot with appropriate micropipettes. 1 μl of a 100 bp DNA Ladder (MBI Fermentas, Hanover, MD, USA) was mixed with 1 μl deionized water and 1 μl bromo phenol blue. Then, 5 μl of this mix was put into usually the first slot as a molecular size marker. The gel was run at 95 V in 0.5X TBE buffer for 50 min. The gel was placed in Gel Doc 2000 (Bio-Rad, Milan, Italy) apparatus and the bands were detected under UV transilluminator.

Table 4 Agarose gel electrophoresis chemicals, buffers and their components and composition concentrations

The marker range used in agarose gel electrophoresis starts from 1000 bp and decreases by 100 bp. The same marker was used in the entire study.

Results

HEK293 cells were created by change of societies of typical human embryonic kidney epithelial cells. As a tentatively changed cell line, HEK293 cells are not an especially decent model for typical cells, malignancy cells, or whatever other sort of cell that is a major question of research. Nonetheless, they are to a great degree simple to work with, being direct to culture.

HUVEC are human umbilical vein endothelial cells that are widely used in research to study vascular endothelium in a cell culture model. Primary human umbilical endothelial cells in culture have a finite proliferative lifespan. They undergo permanent growth arrest, known as replicative senescence.

When replicative senescence is bypassed by transformation with viral oncogene with telomerase reverse transcriptase, HUVEC are immortalized. The HUVE cells we used in our thesis are cell-lines like the HEK293 and HeLa cells (Masters, 2002).

Growth curves were obtained for each cell type and calculated doubling times were calculated to decide for the optimum subculture timing and the cell types nature of proliferation capacity and the nature of the cell whether it is a dividing or non-dividing cell. When working with concepts such as DNA damage, mutation, genomic instability, cell division, mutagenesis, carcinogenesis, and aging, the cell’s nature of division capacity gains utmost importance. The end of log phase is nearly 72 h for HEK cells.

74 for HUVEC cells and 96 h for HeLa cells, so for the time of doubling for each cell type, which is an indication for their proliferation potency, were calculated. We expect to see more change in HEK cells’ DNA, because it replicates faster than the others. Saturation density is the highest for HEK, the second one is HUVEC and the third one is HeLa and the results were all in compromise with the expectations.

Figure 1 is for all cell types’ nearly all passages to see the total DNA variation profile. For HEK cells especially after 9th passage, the changes are more prominent. The arrows show the types of changes observed; the cumulative changes when the passages increase indicate that these are mutations. Loss of bands, appearance of bands, decrease and increase in band intensities all detectable DNA effects have been detected for all cell types, but more prominent in HEK cells, they are epithelial cells that are having the least doubling time, and reaching the highest number two times the other cell types at 72 h, highest saturation density. Some are DNA damages because reverted back in increasing number of passages, some are mutations because were stabilized in increasing number of passages, in long term.

RAPD profiles with OPB7 primer for all cell types’ all subculture DNA extracted. M: DNA size marker. Arrows indicate the most prominent changes of RAPD profiles: loss of bands, appearance of bands, decreases in band intensity and increase in band intensity

In different cell types, the first passages and the last passages are compared in Fig. 2. The stabilization can be easy seen for the last passages. The homogeneity of band profiles is prominent. In all cell types, the remaining bands were quite the same, monomorphic, although the starting profile bands were much more different, polymorphic. The remaining bands at the last passages were having band sizes in between 270 and 470 bp.

Comparable picture of the first and last passages RAPD profiles, circles indicating a pattern homogenization in the last passages no matter what the cell type was

Figure 3 shows the DNA damaging effect of glutaraldehyde which is a DNA–protein cross linker on our different cell types. The first pictures are the Glutaraldehyde exposed cells’ profiles. Profiles are for eight times RAPD-PCR of 21st passages for exposed and 22nd for the glutaraldehyde removed cell samples. For HUVEC and HeLa cells, the DNA effects are reverted back indicating that the changes are DNA damages, but for the more dividing cell, HEK cells, the effects are not reverted back, indication of mutation. The repair mechanisms cannot work properly for HEK cells and this might be an indication that the fast-dividing cells can have difficulties in repair processes which might be causing further pathology.

Glutaraldehyde exposed DNA × 8 RAMD profiles and after removal of glutaraldehyde, the next passages’ DNA × 8 RAMD profiles. The arrows indicate the initial and final profile changes in all cell types. + Glu is for glutaraldehyde added ones and after –glu is after removal of glutaraldehyde from the medium

The effects on DNA should be endogenous, because in cell culture, we aim to have a controlled environment for the cells to continue with the least change. There is a change, a continuous change, but it is detectable in long term rather than changes observed in each passage. The RAPD profiles obtained are in agreement with this. The RAPD-PCR profiles that can detect the intra-specific DNA variation, mosaicism of the individual passage DNA is very homogenous, monomorphic indicating there is a change and if there is a change that is DNA damage.

Discussion

In conclusion, while RAPD and related techniques have been used extensively for diverse studies, application of these techniques has also attracted criticisms with respect to its reproducibility that we have overcame in this thesis by RAPD-PCR approaches. However, it emerges that most of the criticism relates to lack of proper optimization and validation of the techniques in different cell types and species, prior to their applications under in vivo or in vitro conditions. Nevertheless, the RAPD assay and related assays offer great promise especially for the determination of genetic damage under in vivo conditions in wild species and for the evaluation of genomic instability in the process of carcinogenesis. While a large number of new technologies and assays are developing to profile the gene expression pattern either following exposure to environmental contaminants or during the process of malignant development, the RAPD-based techniques offer great promise for future and would continue to complement other new and well-established techniques in population genetics, genotoxicity and carcinogenesis studies and cell-culture work in view of this work.

Data availability

The author has no material discussed in this article.

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Acknowledgements

I would also like to show gratitude to my supervisor, including Ms. I. Irem Uzonur. She died of blood cancer. Her teaching style and enthusiasm for the topic made a strong impression on me and I have always carried positive memories of her classes with me.

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1. Molecular Biology and Genetic, Faculty of Science, Bulent Ecevit University, Zonguldak, Turkey

   Arzu Erol

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Correspondence to Arzu Erol.

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Erol, A. Genomic instability evaluation in different cell line by random amplified polymorphic DNA-PCR analysis. GENOME INSTAB. DIS. (2024). https://doi.org/10.1007/s42764-024-00122-y

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• Received25 December 2023

• Revised08 January 2024

• Accepted15 January 2024

• Published04 March 2024

• DOIhttps://doi.org/10.1007/s42764-024-00122-y

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Keywords

• Genomic instability

• Aging

• RAPD-PCR

• HEK cells

• HUVEC cells

• HeLa cells