Reports-2003-Vol103-N1-Z.A.Karalyan

VIROLOGY

УДК 616.988-092.18

Z. A. Karalyan

Nuclear and nucleolar indices in NIH 3T3 cell line in normal conditions and
under the action of encephalomyocarditis virus

(Submitted by academician K.G. Karageuzyan 23/IV 2002)

   The size and number of the nucleoli are important parameters of a differentiation level and functional condition of a cell. In normal cells nucleolar activity varies in a differentiation course, at the change of a functional condition of cells and at the change of the cell cycle phases [1]. The parameters of the size and nucleolar quantity serve a valuable diagnostic attribute of proliferation speed in transformed cells [2, 3, 4, 5]. In interphase nucleuses nucleolar organizer regions (NOR) of the chromosomes correspond to the fibrillar centres (FC), containing rDNA surrounded by the dense fibrillar and granular components [6, 7]. Decondensed DNA filaments are uniformly distributed in FCs and in transcriptionally active nucleoli they are also present in proximal portion of the dense fibrillar of component surrounding the FCs [8]. Number and the sizes of the FC vary in different cells. The number of FC does not depend on number of chromosomes with active NOR. The sizes of the FC depend on a functional condition of a cells, and from intensity of the transcription of the rDNA [9, 10]. In spite of the fact that in the metaphase chromosomes transcription of the rDNA does not occur, the most part or all NORs is active impregnated by silver [11]. The important parameter in proliferation activity and cells differentiation degree is also the area of nucleuses and nucleoli. So, according to the data [12] the nucleolar areas demonstrate strict dependence from cell population doubling time and consequently from proliferative activity of cells. The dependence of functional condition and sizes of the nucleoli from the proliferative condition of the transformed cells is most obvious [13, 14].
   The purpose of this work was study of dynamics of the various nuclear and nucleolar parameters: nuclear and nucleolar DNA quantities, areas, perimeters of the murine cell line NIH 3T3. The experiments were conducted in normal conditions and under the influence of the murine encephalomyocarditis virus (EMC).
   Materials and methods. We used a NIH 3T3 continuous cell culture of murine fibroblasts which was cultured in Eagle medium with 10% bovine serum. The cell line was received from laboratory U 322 INSERM “Retrovirus et maladies associeйes” Marseilles. A monolayer of the intact cells was used in 48 hours after the passage. Cells was resowed in 10⁵ cell/ml dose. Encephalomyocarditis virus (EMC) was received from Institute of Virology of the NSA of Russia, Moscow. The virus was used at multiplicity of infection 0,1 TCD50 per cell on the 24 hour after resowing cells. The study of parameters began in 24 hours after infection. Infected and intact cells were incubated at the temperature 37⁰C
   The content of DNA by Feulgen staining was defined by computer-equipped microscope-photometer SMP 05 (OPTON). The television method was used on 575 nm wave. In each case 50 - 100 cells were measured. Quantity of DNA was defined in conventional units (C.U.). In nucleuses were simultaneously determined the quantity of DNA, area and perimeter. In the same nucleuses we contoured each nucleoli with the account perinucleolar and intranucleolar chromatin. All statistical analysis were performed with two-tailed Student’s t test in the SPSS version 8.0 computer software package (SPSS, Inc., Chicago, IL).
   Results and Discussion. The data of DNA cytometry in a nucleus and nucleolus, in normal conditions and under the cytopathogenic action of the EMC are given in tables 1 and 2.

Table 1

Parameters of the NIH 3T3 cells in the control

Number of nucleolus in the nucleus	% of the cells in population*	Nucleus			Summarized nucleolus
Number of nucleolus in the nucleus	% of the cells in population*	quantity of DNA (in C.U.)	area	perimeter	quantity of DNA (in C.U.)	Area	perimeter
1	9,8	62,5±7,7	64,5±9,1	19,5±0,7	9,5±0,7	5,7±0,7	6,5±0,7
2	13,2	64,6±20	75,1±14	23,6±3,1	9,8±2,3	8,3±1,5	7,7±2,1
3	25,3	67,0±16	71,1±12	21,1±2,7	11,1±3	8,9±2,1	10,1±2
4	37,5	62,8±11	63,6±9	19,8±1,9	10,8±4	9,3±2,0	12,5±5
5	7,8	62,9±9	83,1±9,6	23,3±2,3	9,4±2,7	10,4±2,3	13,8±2,6**
6 and more	6,4	61,4±4,4	82,2±21	21,5±4,5	9,6±1,1	9,2±2,5	12,7±2,1**

* Without the account the mitosis, dead and nonnucleolar cells
**Significant in comparison with 1 nucleolar cells at 6-nucleolar cells t = 2,80 and at 5 nucleolar cells t = 2,70 (p < 0,01).
The difference between the minimal and maximal measurements of the quantity of DNA in the nucleus and the nucleolus of cells of the line NIH 3T3 in control group (table 1) was less than 10% and less than 15% accordingly. It testifies that the difference between the minimal and maximal measurements of the quantity of DNA in the nucleus and the nucleolus is insignificant in control group. Under the influence of virus the minimal and maximal measurements of the DNA quantity increase in the nucleus approximately 30% and in the nucleolus more than 25% (table 2).

Table 2

Changes of parameters of NIH 3T3 cells under the action of EMC

Number of nucleolus in the nucleus	% of the cells in population*	Nucleus			Summarized nucleolus
Number of nucleolus in the nucleus	% of the cells in population*	quantity of DNA (in C.U.)	area	perimetr	quantity of DNA (in C.U.)	area	perimetr
1	6,5	115±21	92,1±5,6	27,1±2,8	17,1±1	13,5±0,7	7,1±1,4
2	12,2	101±27	66,3±27	19,3±4,9	17,6±3	10,1±3,6	8,3±2,5
3	18,75	124±44	93,5±27	23,5±4,3	20,1±8	12,5±4,7	11,5±3,2
4	35,9	98±30	72,1±14	21,6±4,0	15,9±6	10,8±3,3	12,6±2,9
5	23,9	114±37	83,4±20	23,7±2,8	17,1±5	11,64±3	13,9±2,4***
6 and more	2,75	97±20,6	81,1±19	23,0±3,6	15,4±7	11,1±3,6	14,2±2,2**

* Without the account the mitosis, dead and nonnucleolar cells
** Significant in comparison with 1 nucleolar cells t = 2,72, p < 0,01
*** Significant in comparison with 1 nucleolar cells t = 2,44, p < 0,02
   It necessary to note, that under the influence of acute viral infection the average quantity of DNA in a nucleus of cells of a line NIH 3T3 was significantly increased (control 63,9±9,1 EMC 108,1±14,2 t = 2,62, p < 0,01). Under the action of a virus the quantity of the DNA in nucleolus was also significantly increased (control 10,4±1,3 EMC 17,13±2,2 t = 2,63, p < 0,01).
   In our experiments we obtained only the tendency of increasing of the nuclear area (control 69,8±8,6, EMC 80,4±9,3 t = 0,84). There were not changes of the nuclear perimeters (20,99±2,7- control 22,56±2,9 - infection).
   The one of the parameters of the cells proliferation speed is the change of the nucleolar area (Derenzini et al. 2000). Under the influence of the virus on the NIH 3T3 cells we calculated the tendency of the increasing of the nucleolar area (8,8±0,9 - control, 11,4±1,7 - action EMC t = 1,35).
   Also we investigated the concentration of DNA in a nucleus and nucleolus. Was shown that under the influence of EMC the concentration of DNA in a nucleus was significantly increased (control 0,84±0,097, EMC action 1,31±0,105 t = 3,29, p < 0,01). The changes of the DNA concentration in the nucleolus were insignificant.

Table 3

Nucleolus/nucleus ratio in the control of the NIH 3T3

Number of nucleolus in the nucleus	Nucleolus/nucleus
Number of nucleolus in the nucleus	DNA	area	perimeter
1	0,15±0,01	0,09±0,01	0,33±0,04
2	0,15±0,01	0,11±0,02	0,33±0,01
3	0,16±0,05	0,13±0,2	0,47±0,08
4	0,17±0,06	0,15±0,05	0,63±0,07*
5	0,15±0,05	0,13±0,03	0,59±0,1**
6 and more	0,16±0,06	0,11±0,02	0,59±0,08*

* Significant in comparison with 1 and 2 nucleolar cells t = 2,9, t = 2,8, p < 0,01
** Significant in comparison with 1 nucleolar cells t = 2,04, p < 0,05
As follows from tables 3 and 4 the DNA quantities in the nucleolus/nucleus ratio do not depend from the nucleolus number in the nucleus in the control (average meaning in population 0,161±0,015) and in infection (average meaning in population 0,158±0,02). These data were obtained with taking in account of the percent of each type of cells in the population (tables 1 and 2). The significant difference in the nucleolus/nucleus ratio was absent not only in population, but also in individual cells.

Table 4

Nucleolus/nucleus ratio in the NIH 3T3 cells under the action of the EMC

Number of nucleolus in the nucleus	Nucleolus/nucleus
Number of nucleolus in the nucleus	DNA	area	Perimeter
1	0,15±0,01	0,15±0,002	0,26±0,03
2	0,17±0,01	0,15±0,02	0,43±0,07
3	0,16±0,05	0,13±0,04	0,49±0,1
4	0,16±0,06	0,15±0,04	0,6±0,1*
5	0,15±0,05	0,15±0,04	0,59±0,1*
6 and more	0,16±0,06	0,14±0,05	0,64±0,1**

* Is significant in comparison with 1 nucleolar cells t = 3,23, t = 3,1 p < 0,01
** Is significant in comparison with 1 nucleolar cells t = 3,6, p < 0,001
Under the lytic influence of the EMC the areas in the nucleolus/nucleus ratio of the cell population showed the only tendency to the increasing (0,129±0,02 - control, 0,146±0,02 - experience).
According to the data of tables 3 and 4 there is the only one significant difference between cells NIH 3T3 with the various quantity of nucleolus in nucleus. It is the ratio of the sums of perimeters of the «total» nucleolus to the nucleus. These data demonstrate almost direct dependence with the increase of the nucleolus quantity as in the experiment as in the control.

Figure 1. Distribution of the nucleus by the DNA ploidy (in "c" units) in NIH 3T3 cells

Fig 1 summarizes the changes of the DNA ploidy indices in normal condition and under the influence of EMC. The present results indicate that under the influence of the virus there are the significant changes in ploidy of NIH 3T3 cells, ploidy was increased under the virus influence (2,24 "c" in control 3,79 "c" - EMC infection). Percentage of euploid cells was not changed, (36,3±3,2 in control 36,4±4,2 EMC action). As a result of infection in euploid population the percentage of 2c cells decreases, but the persentage of 4c cells increases. As well in euploid population 8c cells appears.
Our data demonstrate the absence of increase of the total nucleolar DNA with the increase of the number of nucleolus in the nucleus. The quantity of each nucleolar DNA decreases while the nucleolus number in the nucleus increases.

Conclusions

* The quantity of the nucleolus does not depend on the quantity of DNA in a nucleus, as under the influence of virus as in intact cells of NIH 3T3

* The quantity of DNA in the nucleolus in direct proportion with the quantity of DNA in a nucleus as in the experiments as in the intact cells.

* The relation of the quantity of DNA in the system nucleolus/nucleus is stabile state as in the norm as under the influence of a virus.

* The ratio of the sums of the nucleolar perimeters to the nuclear perimeter is the significant factor which increases linearly while the number of the nucleoli in a nucleus increases in the intact cells and under the action of the EMC virus.

* As the quantity of DNA in a nucleus as well the concentration of DNA in a nucleus are significantly increased under the action of the lytic EMC virus.

* There are no changes of the percentage of euploid cells under the influence of the viral infection.

* There is no the increase of the summarized nucleolar DNA while the number of the nucleolus increases in a nucleus of NIH 3T3 cells as under the influence of a virus as in intact cells.

* In the process of the increasing of the number of nucleolus in NIH 3T3 cells there is the decresing the quantity of DNA in each of nucleoli in nucleus.

Cancer Research Center, Yerevan
Institute of molecular biology NSA RA

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