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Estimation of homozygote recessive and heterozygous CDK3 distribution in randomly selected cancer subjects.

INTRODUCTION: Cyclin-dependent kinases (cdk) are serine/threonine protein kinases that play essential roles in the control of cell cycle progression by interacting with a variety of regulators and substrates. In the mammalian cell cycle, the transition from the Go/G1 phase to S phase, in which DNA replication occurs, has been shown to be regulated by cyclin-dependent kinases (cdks). Activities of cdks are controlled by association with cyclins and reversible phosphorylation reactions. An additional level of regulation is provided by inhibitors of cdks. Two families of these inhibitors have been described, those that interact with a wide range of cyclin/cdk complexes, including p21, p27 and p57, and those that only inhibit cdk4 and cdk6, including p15, p16, p18 and p19 [1]. In eukaryotic cells, cell cycle progression is driven by the sequential and periodic activation of cyclin/cdks, and dysregulation of the cell cycle is associated with cancer development [2-4]. Over expression of cdk6 has been observed in lymphomas, leukemias, and melanomas due to chromosomal translocation [5, 6].

Cdks are closely associated with human cancer pathogenesis. Cdk3 is an important regulator of cell cycle. The activity of cdk3 is first observed in early G1 phase [16] and reaches a peak in mid-G1 [7]. A dominant-negative cdk3 was shown to induce G1 arrest, which could not be rescued by cdk2, indicating that cdk3 plays an important role for G1 exit to S entry [8-9]. Recently, cdk3 was found to form a complex with cyclin C and phosphorylate the retinoblastoma protein (pRb) at serine 807/811, which is required for G0-G1 transition [10]. Furthermore, cdk3 seems to be expressed in various normal human tissues and cancer cell lines including glioblastoma and neuroblastoma cells [10-12].

Therefore, since CDK3 polymorphism is in a gene coding for a cell cycle protein and is in an intron region with no known diagnostic value and also the SNP had known allele frequencies in multiple populations, and the two alleles were both common, we have undertaken this project to find out the frequency of polymorphisms in India as preliminary study. The allele frequencies in our population are not available.

MATERIALS AND (METHODS)[11]

Sample Collection: 5ml EDTA Blood sample from 10 healthy volunteers of age group 20 to 35 were taken for the study. DNA was isolated using salting out method. The extracted DNA was subjected to PCR and subsequent restriction digestion to have RFLP patterns using Hpa II.

DNA Extraction by Salting out Method: In brief, 125ul of whole blood wass taken and 125ul of TKM-1 buffer (Tris HCl 10mM, KCl 10mM, Mg[Cl.sub.2] 10mM and EDTA 2mM) was added along with 2-3 drops of Triton--X 100 to remove RBC. Centrifuged at 2700rpm and the supernatant was removed. This step is repeated. Once white pellet is obtained, added 80ul of TKM-2 buffer (Tris HCl 10mM, KCl 10mM, Mg[Cl.sub.2] 10mM, EDTA 2mM and NaCl 0.4M) and 10% 12.5ul of SDS(sodium dodecyl sulphate). This mixture was incubated at 55[degrees]C to dissolve the pellet for 15 min. After dissolution of the pellet freshly prepared 35ul of 5 M NaCl was added and incubated for 10 min. Centrifuged at 12,00rpm to collect the supernatant DNA in fresh tube containing 100% ethanol. After final washing with 70% alcohol, centrifuged to remove the supernatant and air dry the pellet and further dissolved in 100ul TE buffer.

Agarose gel for Electrophoresis: Weighed 0.8g of agarose in 40ml of 1X TBE Buffer in a micro-oven and added 4 [micro]L of Ethidium Bromide (EtBr) as soon as it is removed from oven and mixed to make it uniform. Pour it in the prepared gel casting plate and allow it to solidify. After solidification remove the comb and place it in gel tank containing TBE buffer. Place loading place towards negative cord plug and the samples are loaded into the wells along with ladder. Voltage was set to 100V and the gel was inspected until the loading buffer band reaches the near to the opposite end. After running the gel completely, bands were observed on UV Transilluminator.

PCR Setup: The PCR is set up with 20pM of forward and reverse primers. The reaction is setup in 20ul of reaction volume with final concentration of 50mM KCl, 10mM Tris, and 200mM dNTP's and 1.5mM Mg[Cl.sub.2]. Reaction were setup under the cycling parameters 940[micro]C for 4min, 940[micro]C for 1min, 55[micro]C for 1min, 72[micro]C for 1min, 72[micro]C for 10 min. The number of PCR cycles was 35.

Primers: The resultant PCR product was loaded in 2% Agarose Gel Electrophoresis the expected band size is 308bp which is run along with the DNA ladder. From the resultant PCR product 5ul of the sample is used for the Restriction Digestion.

RESULTS AND DISCUSSION: Table 1: Restriction Digestion of the sample was done using Hind III Restriction Enzyme. Reaction is set up for 20 [micro]l of reaction volume with the final concentration of 10mM Tris-HCl, 10mM and enzyme 0.4 [micro]g/100 [micro]l. Distributed 10 [micro]l of reaction mix in 10 tubes as shown below in table. The expected band size after digestion is 209bp and 99bp.

Sl/No   Sample ID      Restriction      PCR Product
                    digestion Mixture

1       SD3 166r       10 [micro]l      10 [micro]l
2       SD2 157r       10 [micro]l      10 [micro]l
3       SD3 294r       10 [micro]l      10 [micro]l
4       SD4 304r       10 [micro]l      10 [micro]l
5       SD2 166r       10 [micro]l      10 [micro]l
6       SD2 274r       10 [micro]l      10 [micro]l
7        SD2 56r       10 [micro]l      10 [micro]l
8       SD1 185r       10 [micro]l      10 [micro]l
9       SD1 119r       10 [micro]l      10 [micro]l
10      SD2 226r       10 [micro]l      10 [micro]l


Based on the banding patterns observed in gel image 1 and gel image 2 the samples are interpreted as Homozygote dominant, Homozygote recessive and Heterozygous. The RFLP results are shown in Image 1 & 2. All samples were successfully amplified except one. In sample number SD2 274 has undigested parental band is observed. Since the 306bp band is faint when compared to rest of the bands, it should be undigested band. From the population we have selected the frequency of Homozygote recessive is 55.5%, heterozygote is 44.4% as mentioned in the Table 2.

In figure 1, lane 2 showed undigested product and lane 3 showed digested product of sample SD3 166. Digestion with HPA 2 restriction enzyme produced 2 bands 209 bp and 99 bp indicating both alleles have G polymorphs which are true with sample number 157 of which undigested and digested products are shown in lane 4 and 5 respectively. Lanes 9 and 10 indicates undigested and digested products of SD4 304 no Amplicon. Corresponding undigested and digested products are nor seen. Probably it's due to isolation of DNA was faulty. Lanes 11 and 12 show the undigested and digested products of SD2 166 sample indicate 308 bp, 209 bp and 99 bp respectively indicating 2 alleles have G in place of A.

In figure 2, lane 2 showed undigested product and lane 3 showed digested product of sample SD3 274r. Digestion with HPA 2 restriction enzyme produced 2 bands 209 bp and 99 bp indicating both alleles have G polymorphs which are true with sample number SD2 56 of which undigested and digested products are shown in lane 4 and 5 respectively. In sample SD2 274 a faint 308 bp is seen, it is probably undigested product. Lanes 8 and 9 indicates undigested and digested products of SD1 119. Lanes 10 and 11 show the undigested and digested products of SD2 226 sample indicate 308 bp, 209 bp and 99 bp respectively indicating 2 alleles have G in place of A.

The nucleotide sequence selected for PCR: 5' AAGGGCGTGT AGCACAGCAT AAAGACAGAG CTAACTCAAT GAGCGCCACT TTCACAGGGA AGATAAATAC TGCACTTATC CTGGGGGAGG CTTCCAGGTT GAACAATCAG TATACCCAAG CCAGTTGTGT ACAAAGGTCA GGAAAGAGAC CCTGGCCTTG GACTCAGAAA GTGCCAGGGT TATGTAAGAG GCTGGCTGAT GAGGGGAAAC TGTAGTCGGA GCAGCAGCTG GAGCCCACAT GCACCTACCA TGAGCAGGTC CCTGCCCTCT GGCTCCAGAT TGGGCACAAT CTCTTCCAGT CCCTTCCTGG T3' the nucleotide position 96 could be A or G. The site which the restriction enzyme Hpa II recognizes is 5' CCGG 3'. Hpa II cuts the sugar phosphate backbone between the two C's at the 5' end of the sequence: 5' C|CGG 3'.

REFERENCES:

[1.] Malumbres M, Barbacid M. To cycle or not to cycle: a critical decision in cancer. Nat Rev Cancer 2001; 1: 222-31.

[2.] de Carcer G, de Castro IP, Malumbres M. Targeting cell cycle kinases for cancer therapy. Curr Med Chem. 2007;14:969-85.

[3.] Wolfel T, Hauer M, Schneider J, et al. A p16INK4a insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 1995; 269: 1281-4.

[4.] Malumbres M, Barbacid M. Mammalian cyclin dependent kinases. Trends Biochem Sci 2005; 30:630-41.

[5.] Corcoran MM, Mould SJ, Orchard JA, et al. Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations. Oncogene 1999; 18:6271-7.

[6.] Hayette S, Tigaud I, Callet-Bauchu E, et al. In B-cell chronic lymphocytic leukemia's, 7q21 translocations lead to over expression of the CDK6 gene. Blood 2003; 102:1549-50.

[7.] Van den Heuvel S, Harlow E. Distinct roles for cyclin dependent kinases in cell cycle control. Science 1993; 262:2050-4.

[8.] Hofmann F, Livingston DM. Differential effects of cdk2 and cdk3 on the control of pRb and E2F function during G1 exit. Genes Dev 1996; 10:851-61.

[9.] Ren S, Rollins BJ. Cyclin C/cdk3 promotes Rb dependent G0 exit. Cell 2004; 117:239-51.

[10.] Meyerson M, Enders GH, Wu CL, et al. A family of human cdc2-related protein kinases. EMBO J 1992; 11: 2909-17.

[11.] Schang LM, Bantly A, Schaffer PA. Explant-induced reactivation of herpes simplex virus occurs in neurons expressing nuclear cdk2 and cdk4. J Virol 2002; 76:7724-35.

[12.] Bullrich F, MacLachlan TK, Sang N, et al. Chromosomal mapping of members of the cdc2 family of protein kinases, cdk3, cdk6, PISSLRE, and PITALRE, and a cdk inhibitor, p27Kip1, to regions involved in human cancer. Cancer Res 1995; 55:1199-205.

K.P.S. Adinarayana (1), Rushinadha Rao Kakara (2)

PARTICULARS OF CONTRIBUTORS:

(1.) Associate Professor, Department of Anatomy, Andhra Medical College, Vishakhapatnam, Andhra Pradesh, India.

(2.) Research Associate, Central Institute of Fisheries Technology, Indian Council of Agricultural Research, AU Po; Vishakhapatnam, Andhra Pradesh, India.

NAME ADDRESS EMAIL ID OF THE CORRESPONDING AUTHOR:

Dr. K.P.S Adinarayana, Associate Professor, Department of Anatomy, Andhra Medical College, Vishakhapatnam, Andhra Pradesh, India.

Email--kpsanarayana@rediffmail.com

Date of Submission: 25/10/2013.

Date of Peer Review: 26/10/2013.

Date of Acceptance: 30/10/2013.

Date of Publishing: 06/11/2013

Table 2: The CDK3 RFLP--Interpretation

Sl No   Sample ID     Bands detected (bp)            Results

1       SD3 166r        209 bp and 99 bp       Homozygous Recessive
2       SD2 157r        209 bp and 99 bp       Homozygous Recessive
3       SD3 294r    308 bp, 209 bp and 99 bp       Heterozygous
4        SD4304r                                       N/A
5        SD2166r        209 bp and 99 bp       Homozygous Recessive
6       SD2 274r        209 bp and 99 bp       Homozygous Recessive
7        SD2 56r        209 bp and 99 bp       Homozygous Recessive
8       SD1 185r    308 bp, 209 bp and 99 bp       Heterozygous
9        SD1119r    308 bp, 209 bp and 99 bp       Heterozygous
10      SD2 226r    308 bp, 209 bp and 99 bp       Heterozygous
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Title Annotation:ORIGINAL ARTICLE
Author:Adinarayana, K.P.S.; Kakara, Rushinadha Rao
Publication:Journal of Evolution of Medical and Dental Sciences
Article Type:Report
Geographic Code:9INDI
Date:Nov 11, 2013
Words:1896
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