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As the cancer is the most distressing problem, the researches found some genetic alteration and errors in mitosis. The main aim of this laboration was to study the chromosome morphologically and mitotic index of mitomycin C as well. The mitomycin C treated and untreated NUT-97 cells used along with a drug colcemid to arrest the chromosomes in metaphase. Colcemide prevents microtubule formation which leads to enhance the cell ability respect to the anaphase. Giemsa staing agent used to highlight and visualize bilogical structure under the microscope. Ploidy of cell found to be increased whereas triploid and tetraploid were counted under the microscope. Increased ploidy was found in cancer cell as presence of the tumorigenesis.
Cancer is known as the most prominent fatal disease in the world and it leads to the health problem as well. It is importent to study the molecular mechanism involved in cancer and cure by the specific treatment. About 60% of cancer patients having malignant tumor. Somatic cells such as G1, G2, S and M undergoes to the DNA replication, cytokinesis and karyokinesis results into two daugher cells (Rasnick, 2000). Controll cell growth is associated to the balance between stimulatory and inhibitory signals for growth. Stimulatory signals increased by genetic alteration in somatic cells leads to genetic instability which makes the cell proliferative about the selective growth advantage. Thus it leads to many complications such as cancer (Malmon, 1964).
In somatic cell, 46 chromosomes are present in 23 pairs and is diploid. Whereas, 23 characteristics chromosomes seems to be in quadruplicate and triplicate in cancer cells. Solid tumor of cancer cells consist the increasing number of 60-90 chromosomes. Cell consisting 23 chromosomes in a pair is called as diploid cell and the cells having abnormal chromosomal complement known as aneuploidy cell. The imbalance in complement chromosomes resulting many complications such as cancer. Aneuploid cell formed due to the errors in mitosis. Radiations is also a cause of formation of the aneuploidy cell. About 90% cancer cells are aneuploidy. It is suggested that almost all type of cancer cells are aneuploidy (Rasnick, 2000).
Chromosomal stain (A Giemsa staining)
Giemsa staining is used to visualise the metaphases under the microscope. Acetocarmine, haematoylin and gentian violet can be used. This technique is designed to differentiate the morphology of nuclear and cytoplasmic contents. It is also known as DNA binding stain.
Mitomycin-C treated and untreated NUT-97 (rat uterine tumor cells) were observed under the microscope in respect to the presence of chromosomal aberrations. Mitomycin-C is a antibiotic derived from Streptomyce caespitosus having a anti-tumor activity which can prevent the DNA replication. It is also known as DNA damaging agent and it produces chromosomal aberrations while treated with NUT-97 (Rasnick 2000, Malmon 1964).
Aim of the present laboration was to prepare chromosomes from NUT-97 mitotic cell, perform a Giemsa staining and to study the difference between chromosome morphologically of Mytomycin-C treated and untreated cells; And to analyze the ploidy of the cells by counting the mitotic index.
Material and methods
The procedure for present laboratory experiment was performed as per the protocol given in laboratory manual of tumor biology course fall 2010. the laboratory experiment was divided in two steps. Step first was consisting the chromosomal preparation and the second included chromosomal staining. Whereas, the NUT-97 cells were prepared by the supervisors.
Preparation of chromosomes (Day 1)
NUT-97 cells were treated with 10 Î¼g/ml of Mitomycin-C. 3 drops (35Âµl) of colcemid was added (to stop the growth of cells in metaphase) and incubated at 370C for 35 minutes. Then it mitotic shake off was done. After the mitotic shake off, centrifugation was perfomed at 1000rpm for 5 minutes, supernatant was discarded. Hypotonic treatment was performed, in which 4-5 ml KCL was added. Washed with mixture of methanol and acetic acid (ratio 9:1) and the cells were spinded again at 1000 rpm for another 5 minutes, supernatant was discarded. 4 ml of methanol and acetic acid (ratio 9:1) was added to fixed the cells. It was spinded again for 5 minutes at 1000 rpm, supernatant was discarded. Same procedure was repeated by using 5:1 and 3:1 ratio of methanol and acetic acid.
Chromosomal staining (Day 2)
15-20 Âµl (a drop) of cell suspension of Mitomycin-C treated and untreated was taken on slide, air dried and it was stained by 5% Giemsa staining solution. The slide was dipped into the staining solution for 10-15 minutes, air dried and fixed with cover slip. Slide was observed under the light microscope using 40X and 100X magnification.
Result and Discussion
In this experiment NUT97 cells (culture of rat uterine tumor) were treated with Mitomycin-C (concentration 10Î¼g/ml) and analyzed in respect to the average number of chromosomes (ranges from 30-60) for more than 20 cells.
Chromosomal preparation was followed by using NUT97 (Mitomycin-C treated) and NUT97 (Mitomycin-C untreated). Metaphase stage of the cell is much important to know about the structural chromosomal aberrations and aneuploidies as well. Whereas, use of colcemide (Figure A) resulted to increasing the large number of metaphases in the cell culture. And these colcemide treated cell culture preparation were stained by GIEMSA reagent. Furthermore laboratory bench light microscopes were used along with Fluorescence microscopy to observe the stained chromosomal preparation.
Figure A: Colcemide treated NUT97 preparation
Mitotic Index was counted followed by counting the total number of nucleus and metaphases which is focused in Figure A. Mitotic Index is the ratio of number of metaphases to the total number of nucleus focused in the specific area. And the obtained results are shown below in table 1.
Unstained chromosomal preparation could not visualize metaphases under the Laboratory bench light microscope. There are 27-32 average numbers of chromosomes were found in Fluorescence microscopy by performed GIEMSA stain (Figure B). (Table no.1)
Figure B : Metaphase stage visualized by GIEMSA staining.
In the other hand, the chromosomal preparation was treated by mitomysin C (Figure C). According to above figure C, the more exposure to the mitomysin C produces inhibition of the metaphase which result to no metaphases were found under the microscope.
Figure C : Mitomysin C treated chromosomal preparation
According to the above (Table no. 1) observation, the average number of chromosomal count in metaphases got slightly less than the normal count. This error might have been occurred due to more dilution of chromosome preparation during the experiment.
Table No. 1: Number of chromosomes counted in few groups of metaphase for Mytomicin-C treated and untreated NUT97.
Number of Metaphases Group
Number of Chromosomes counted
The total number of chromosomes were counted present in each metaphase. Whereas, 21 sets of chromosomes are there in rat uterine cells. Ploidy is the number of sets of chromosomes present in biological cells and it was calculated by dividing the number of chromosomes by sets of chromosomes present in each of the biological rat cells, which are 21.
Mitotic Index = The total number of cells in metaphase
Total number of cells counted
Table No. 2: Average number of chromosomes counted in few groups of metaphase and total number of nucleus counted.
Average number of Chromosomal count in metaphases group
Number of nucleus counted
From the obtained result, the value of mitotic index seems to be varying than the expected value. Error in counting might be subjected to the less count of metaphases and the nucleus as well. It may leads to varying in the value of mitotic index. These errors can be evaded by making a proper chromosomal dilution and counting of chromosomes several times.