The Effect of cdna on Tumor Cells Growth on Nude Mice

The Effect of cdna on Tumor Cells Growth on Nude Mice Yiming Ding Department of Electric and Computer Engineering Portland State University, OR Email: dym@cecs.pdx.edu This project is assigned in the class Learning from Data, offered in Winter 2002. 1

Abstract: CSK homologous kinase (CHK) is a negative growth regulator of human breast cancer. This study shows that the recombinant DNA of CHK affects breast cancer growth and DNA of CHK and DNA of CSK affect ovarian cancer growth on nude mice. The data are processed statistically and demonstrate the cdnas posses inhibitory effect on tumor growth. cdna is strong, cloned copies of otherwise fragile mrna - the essential messenger element of the genes in the DNA which help in the coding of proteins. Recombinant DNA refers to DNA, which has been altered by joining genetic material from two different sources. It usually involves putting a gene from one organism into the genome of a different organism, generally of a different species. Keywords: CHK, nude mice and cancer. Introduction CSK homologous kinase (CHK) is a novel negative growth regulator of human breast cancer. In previous studies, it is well known that the CHK(DNA) possess inhibitory effect on breast cancer cells growth in vivo as well as in vitro studies. Also it is known that recombination DNA CHK shows the same effect in vitro studies. Figure 1 shows the in vivo study of CHK effect on the breast cancer MCF-7 cells. Four subtype MCF-7 cells were injected into nude mice with same amount. Type one: wild-type MCF-7 cells. Type two: MCF-7 cells transfected with pcdna-iii(vector). Type three: MCF-7 cells transfected with mutant CHK gene. Type four: MCF-7 cells transfected with CHK gene We could see that the tumors with CHK gene disappeared after 45 days and it was confirmed by dissection. The tumors with mutant CHK gene were much smaller than tumors of wild type MCF-7 and tumors with vector alone. 2

MCF-7 Cells Growth Curve on Nude Mice 250 Volume (cubit mm) 200 150 100 50 0 day 10 day15 day 23 day 31 day 37 day 45 day 52 Day wild type vector CHK mt CHK Fig. 1 CHK effects on MCF-7 cells growth in vivo. Two experiments were performed in Dr. Hava Avraham s Lab, Department of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston. The purpose was to observe the tumor growth on nude mice in the conditions of cdna, Chitosan(a compound protecting DNA from digestion of DNAse in the blood), or combinations injected into mice. We try to answer the question: Do the cdnas have an inhibitory effect on tumor growth in vivo? in our research. Methodology Animal model: 3 week-5 week old female nude mice were injected with pre-cultured breast cancer cells, under the skin of lower abdomen, 6 5 10 cells per each mouse. The cells were resuspended in 300 µ l Phosphate Buffer Saline and Matrigel with ratio of 1:1. In the 3

first experiment breast cancer MDA-MB-231 cells were injected to the mice while the second experiment ovarian cancer SK-OV-3 cells were injected. Grouping: Randomly group the mice into 4 to 6 groups, 4 to 5 mice for each group (basically the number of mice in each group is equal in each experiment). Measurement: After 7-10 days injection of tumor cells, the injection sites became solid, we measured the sizes of the masses in three dimensions (length, width and depth), and followed up to the end of the experiment (at least three weeks). Measurements were taken twice a week. DNA and Chitosan preparation: 500 µ g DNA were dissolved in 200 µ l 500mM Sulfate Sodium. Chitossan were dissolved in Sulfate Sodium with 0.2% concentration. Treatment: When the tumor becomes solid, we started injection of same dose (100 µ g ) cdnas, Chitosan or combinations twice a week, except the control group. In the first experiment, we injected Chitosan, Chitosan plus pcdnaiii(vector), Chitosan plus CHK or Chitosan plus mutant CHK. In the second experiment, we injected Chitosan, Chitosan plus CHK, Chitosan plus mutant CHK, Chitosan plus CSK or Chitosan plus mutant CSK. We also gave injection of phosphate buffer saline to the mice in the control group. Experiment results: In the first experiment, after half month of the treatment, the volume of tumors in the control group was much larger than that in other groups(fig. 2). In the second experiment, after three weeks of treatment there were some differences in volume of tumors between the groups(fig. 3). 4

Exp. 1: cdna Effects on MDA-MB-231 Tumor Growth on Nude Mice Volume(Cubic mm) 400 350 300 250 200 150 100 50 0 Day 3 Day 6 Day 10 Day 13 Day 17 Day 20 Day 24 control Chitosan pcdna3+chitosan CHK+Chitosan CHK(mt)+Chitosan Fig. 2 Experiment 1:cDNA s Effects on MDA-NA-231 Cells growth Exp2 cdna Treatment Effects on SK-OC-3 Cells Growth on Nude Mice Volume(Cubic mm) 1000 900 800 700 600 500 400 300 200 100 0 DAY 23 DAY 29 DAY 37 DAY 44 PBS Chitosan CHK+Chitosan CHK(mt)+Chitosan CSK+Chitosan CSK(mt)+Chitosan Fig. 3 Experiment 2:cDNA s Effects on SK-OC-3 Cells growth Statistic Analysis and Discussion 5

Due to the purpose of the study, the interested parameter was Growth Rate of Tumor, Y2 Y1 defined as the increasing volume of mass in certain period ( Rate = ). X X Y1: Volume at the beginning Y2: Volume at the end X1: Starting time (day) X2: Ending time (day) The first step is to calculate the growth rate of tumors from raw data in each experiment. Table 1 and table 2 show the results. Animal ID Growth rate Control 1 32.29 2 12.14 3 12.29 4 6.76 5 7.52 Chitosan 1 6.76 2 7.81 3 0 4 2.57 5-1.33 Chitosan+Vector 1-1.62 2 0.33 3 5.71 CHK+Chitosan 1 1.9 2 1.29 3 6.29 4 6 5 0.71 CHK(mt)+Chitosan 1 1.24 2 1.57 3 1.05 4 5.05 5 5 Table 1. Growth rates of tumors in experiment 1 2 1 6

Animal ID Growth rate Control 1 53.14 2 56.29 3 12 4 33.29 Chitosan 1 26.29 2 41.14 3 14.29 4 11.67 CHK+Chitosan 1 3.62 2 5.81 3 17.19 4 14.10 CHK(mt)+Chitosan 1 10.29 2 6.86 3 24.29 4 5.48 CSK+Chitosan 1 3.14 2 10.86 3 13.71 4 9.52 CSK(mt)+Chitosan 1 14.14 2 7.62 3 6.14 4 17.24 Table 2. Growth rates of tumors in experiment 2 The second step is to test the data with lilletest, the histograms show that the growth rates are not normal distributed (fig.4 and fig.5). The third step is to compare the growth rate between control group and other groups by pair using Rank-Sum Test (Non- parametric method). This method does not require large number of samples in each group. There were two hypothesis: H0: The growth rate of the tumor in control group(µ 1 ) is equal to the rate in cdna groups(µ 2 ) at 0.05 significant level. 7

8-5 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 7 8 9 Fig. 4 Growth rate distribution in experiment 1 0 10 20 30 40 50 60 0 1 2 3 4 5 6 Fig. 5 Growth rate distribution in experiment 2

H1: The growth rate of the tumor in control group (µ 1 ) is greater than the rate in cdna groups(µ 2 ) at 0.05 significant level. Table 3 shows the way of Rank-Sum Test To Test H 0 Versus H 1 Compute µ 1 = µ 2 µ 1 < µ 2 µ 1 > µ 2 µ 1 µ 2 µ 1 µ 2 µ Table 3.Rank-Sum Test Decision criteria of Rank-Sum test: If the observed value µ, µ1, or µ2 is less than or equal to the table critical value, we reject H0. This report computed µ 2 and chose one tailed at 0.05 significant level. Table 4 and table 5 show the outcomes of the Rank-Sum test of the two experiments. µ 2 Critical Value Control/Chitosan 2.5 4 Control/Chitosan+Vector 0 4 Control/CHK+Chitosan 0 4 Control/CHK(mt)+Chitosan 0 4 Table 4. Rank-Sum test outcome of experiment 1 µ 2 Critical Value Control/Chitosan 4 1 Control/CHK+Chitosan 2 1 Control/CHK(mt)+Chitosan 1 1 Control/CSK+Chitosan 1 1 Control/CSK(mt)+Chitosan 2 1 Table 5. Rank-Sum test outcome of experiment 2 In the first experiment, the result of Rank-Sum test rejects H 0 in all comparison pairs. In the second experiment, the result of Rank-Sum test rejects H 0 in comparison pairs of Control 9

versus mutant CHK plus Chitosan and Control versus CSK plus Chitosan; but fails to reject H 0 in other comparison pairs. Conclusion Based on the statistic analysis on the experiments, we can conclude: 1. Both CHK and mutant CHK can inhibit MDA-MB-231 cells growth on nude mice. 2. CHK and mutant CSK do not inhibit SK-OV-3 cells growth on nude mice, but CSK and mutant CHK do. Acknowledgement Thank Dr. Hava Avraham s support and Yigong Fu s contribution to this project. Thank Dr. James McName s for his guides to the statistics analysis 10