INTERACTION OF TUMOR CELLS AND IMMUNE SYSTEM: INTERINDUCTION OF APOPTOSIS BETWEEN TUMOR HEPATOCYTES AND SPLENOCYTES

L.B. Ginkul, S.A. Alexandrova, I.N.Shvemberger
Institute of Cytology Russian Academy of Sciences
 St. Petersburg, Russia
1.1.   Introduction
L.B.Ginkul    lbgin@mail.cytspb.rssi.ru
Normal development and functioning of all parts of an organism as well as response of the organism to adverse factors (cell malignant transformation, infections, and various other damages) are provided by a complex of biological systems in all organisms from unicellular to the high mammals. In the course of evolution, such systems become more complex and more numerous. But their basic function is to maintain homeostasis at the cellular, tissue and organism levels. The immune system plays the key role in the high animals, but there is always an active interaction between elements of the immune system and other organism elements. One of powerful systems protecting an organism from various damages is apoptosis. Apoptosis is the genetically programmed cell death; it is involved in embryogenesis and normal functioning of organs and tissues as well as in various pathologies, including tumor growth [Filchenkov and Stoika 1996; Hug 1997; O`Connor 1998; Rinkenberger and Korsmeyer 1997]. Therefore, investigation of apoptosis in malignant cells needs a combined approach. The goal of this work was to study interrelations between quantity of genetic rearrangements in hepatoma MH-22a clonal lines revealed by RAPD-PCR and their capability for interinduction of apoptosis at their co-cultivation with singenic splenocytes. A possible correlation between the quantity of revealed genetic rearrangements in clonal lines of tumor hepatocytes and their ability to differentiate in vivo during the growth in the eye anterior chamber was also examined.

1.2. The experiments arrangement
The study of interaction between tumor cells and splenocytes was carried out on the mouse hepatoma MH 22a cell line and its clonal lines (Fig.1). 

Figure 1. Production of clonal lines from hepatoma MH-22a cell line.

1.2.1. Interinduction of apoptosis
To induce apoptosis, splenocytes from spleen of the 6-9-week old singenic C3HA mice were used. All mice were sacrificed after previous narcosis. For interinduction of apoptosis between hepatocytes and splenocytes they were mixed at a 50:1 ratio and co-cultivated for 18 h. As a control, splenocytes and hepatocytes were also cultivated separately for 18 h. After induction of apoptosis, low molecular DNA was isolated by a modified Hirt’s method [Vikhanskaya 1994]. For detection of the oligonucleosomal DNA fragmentation, an electrophoresis in 2% agarose gel was applied. The level of apoptosis in hepatocytes was also evaluated by the clonal survival test. Approximately 250 tumor hepatocytes were placed in Petri dishes after their co-cultivation with splenocytes. The number of clones was counted, when they reached diameter of 1-2 mm. Tumor hepatocytes incubated without splenocytes were used as a control.

Figure 2. Scheme of experiments for interinduction of apoptosis between hepatocytes MH-22a and splenocytes, and revealing of apoptosis by electrophoresis.

1.2.2. Induction of tumor cell differentiation
To assess capability of MH-22a and its clonal lines for differentiation, their cells were transplanted into subcutaneous connective tissue and into the eye anterior chamber.

1.2.3. The revealing of genetic heterogeneity
The genetic heterogeneity in the cell population of MH-22a hepatocytes was detected by RAPD-PCR. As primers for RAPD-PCR, three synthetic decanucleotides were used. Products of RAPD-PCR were separated electrophoretically on 2% agarose gel in Tris-borate buffer. DNA fragments were stained with ethidium bromide. RAPD PCR probes obtained with primer but without DNA and with DNA without primer were used as controls.

1.2.4. Histology
For routine histology, tumors were fixed with 10% formalin, passed through different concentrations of ethanol and embedded in paraffin. Sections (5 μm) were stained with hematoxylin-eosin.

1.3. Interinduction of apoptosis between tumor hepatocytes and normal splenocytes

1.3.1. Apoptosis in tumor clonal lines and splenocytes
The interinduction of apoptosis between tumor hepatocytes and singenic splenocytes was studied on the clonal lines of hepatoma MH-22a. It was found that both the basic population of tumor hepatocytes and all studied clonal lines were able to induce apoptosis in splenocytes, although intensity of apoptosis in splenocytes was different. Meanwhile, splenocytes induced apoptosis in tumor hepatocytes only in the basic population and in three out of five examined clonal lines, 22/5, 22/7, and 22/8 (Table). In the clonal line 22/7, apoptosis was detected only by the method of clonal survival, but not by electrophoresis of low molecular DNA fractions. In the clonal lines 22/4 and 22/6, both methods used for apoptosis detection produced negative results. Clonal survival in these clonal lines was 99% (Fig. 3). 

                 e   c       e    c        e    c       e    c         e   c  
                 Basic        Clonal       Clonal        Clonal       Clonal
                population  line 22/4    line 22/5    line 22/6    line 22/7

Figure 3. Clonal survival MH-22a cells after co-incubation with syngenic splenocytes. e – experiment, c – control.

1.3.2. Genetic polymorphism in hepatoma cell line MH-22a and its clonal lines
In the basic cell population of hepatoma MH-22a and in its five clonal lines, genetic variability of DNA was studied with three random primers – 447, 452, and 453. In each case, the spectra of amplified DNA fragments consisted of 14-17 DNA fragments and differed in their molecular weight range: for primer 447 – from 300 to 3000 bp, for primer 452 – from 900 to 3000 bp, and for primer 453 – from 450 to 1300 bp (Fig. 4). Each spectrum consisted of 14-17 amplified DNA fragments. Analysis of the amplified DNA fragments spectra revealed differences between the fragments with respect to their variability: the appearance and disappearance of new fragments, a shift of the fragments, and an increase or decrease of fluorescence level of the DNA fragments. According to the data obtained with use of all three primers both in vitro and in vivo, the most variable DNA fragments were 500, 750, 900, and 1400 bp.

Figure 4. Electrophoregrams of amplified DNA fragments obtained from cells of the basic population of tumor hepatocytes of MH-22a and its clonal lines in vitro.
a – primer 447; b – primer 452; c – primer 453.
1 – liver of C3HA mice; 2 – basic population of MH-22a; 3–7 – clonal lines: 3 – 22/4, 4 – 22/5, 5 – 22/6, 6 – 22/7, and 7 – 22/8, M - DNA ladder 100 bp.

	Although similar results were obtained using all three primers, the most pronounced changes were observed in spectra of the amplified DNA fragments with the primer 447. To analyze genetic variability, the amplified DNA fragment spectra of clonal lines were compared in vitro with the amplified DNA fragment spectrum of the basic cell population MH-22a. It was shown that the clonal lines differed by the amount of genetic rearrangements from the basic population and from each other (Table). The genetic variability in the basic population of MH-22a and its clonal lines were also detected in vivo at their transplantation into the eye anterior chamber and subcutaneous connective tissue. To detect genetic variability in vivo, spectra of amplified DNA fragments in the eye anterior chamber and subcutaneous connective tissue transplants were compared with the amplified DNA fragment spectra of the basic cell population of hepatocytes and clonal lines in vitro. Under these conditions the genetic variability was shown to increase both in the basic population of tumor hepatocytes and in clonal lines. The only exception was the clonal line 22/5, in which no changes were detected (Table). It was revealed that the level of genetic 

Table. Results of experiments for complex studying genetic variability of tumor hepatocytes MH-22a, their capacity for differentiation at growth in the EAC and for interinduction of apoptosis between hepatocytes and syngenic splenocytes at their combine cultivation

MH-22a cell 
line	Genetic variability of hepatocytes	Differentiation of hepatocytes grown in	Apoptosis 
revealed in	
	  in
vitro	in vivo	

SCT	

EAC	    hepatocytes	spleno-
cytes
EP	
		SCT	EAC			CS	EP		
Basic
population	
	
++	
+	
   -	
   +	
+	
+	
+	
Clonal lines:
22/4	
+++	
++	
+	
   -	
   -	
-	
-	
+	
22/5	-	-	-	   -	   +	+	+	+	
22/6	-	++	+++	   -	   -	-	-	+	
22/7	++	nd	++	 nd	   +	+	-	+	
22/8
	+	+	+	   -	   +	nd	+	+	

In vitro – DNA polymorphism in clonal lines of tumor hepatocytes estimated in comparison with DNA polymorphism of basic population of MH-22a taken as a zero.
In vivo - DNA polymorphism in tumor hepatocytes at their growth in SCT and EAC estimated in comparison with their DNA polymorphism at growth in vitro taken as a zero. 
CS – clonal surviving; EF – electrophoresis of low molecular fractions of DNA; nd – non data.

rearrangements in the basic population and the clonal line 22/4 decreased at the growth in eye anterior chamber in comparison with that in subcutaneous connective tissue; it did not differ in two clonal lines, 22/5 and 22/8, and increased in the clonal line 22/6(Fig. 4). Both the basic hepatocyte population and hepatocytes of three clonal lines (22/5, 22/7, and 22/8) were found to be able to differentiate at the growth in eye anterior chamber. Signs of cytotypic differentiation (large cells, structured nuclei, the light eosinophilic cytoplasm, a small number of mitoses) and moderate signs of histotypic differentiation (formation of clusters of differentiated hepatocytes surrounded by thin collagen fibers) were observed (Fig. 5).

Figure 5. Transplants of hepatoma MH-22a in eye anterior chamber.
a – basic population of MH-22a; b – nondifferentiated clonal line; c - differentiated clonal line. 
a, b – hematoxilin-eosin; c – Van-Gizon; x400.

1.3.3.	Discussion 
It has been established that tumor progression, dream state of cells or spontaneous regression of tumor take place in neoplastic growth [Shvemberger 1986, 1987; Steinbach 2002]. Much less is known how these states of tumor cells depend on the instability of their genome and ability of tumor cells to differentiate. Study of apoptosis at tumorigenesis has shown its great role at different stages of the disease, beginning from the appearance of the first atypical cells. However, some issues still are poorly studied, particularly the role of biological peculiarities of tumor cells in the interinduction of apoptosis between cancer cells and lymphocytes [Pinkoski and Green 2000; Scaffidi et al 1999].
The study of heterogeneity of the hepatoma cell line MH-22a by clonal analysis has allowed us to suggest a correlation between the genetic variability, capability for differentiation at the growth in eye anterior chamber, ability to induce apoptosis in splenocytes by tumor hepatocytes, and the resistance of tumor hepatocytes to splenocyte-induced apoptosis. The data obtained may be summarized as follows:
Clonal lines 22/4 and 22/6 are characterized by a high level of genetic variability; lack of differentiation at growth in eye anterior chamber; resistance to the apoptosis-induced by splenocytes; capability for the apoptosis induction in splenocytes.
Basic population, clonal lines 22/5, 22/7, and 22/8, are characterized by a low level of genetic variability; capability for differentiation at growth in eye anterior chamber; interinduction of apoptosis between tumor hepatocytes and splenocytes. These data indicate the general role of the genetic variability level in the studied features of tumor cells. RAPD-PCR applied for revealing of the DNA-polymorphism may be used as a non-specific probe that might characterize rearrangements in genome occupying some hundreds or thousands of nucleotides [Shvemberger and Alexandrova 2000]. Hence, it may be concluded that even non-specific genome disturbances affected such biological characters of tumor cells as their capabilities for differentiation at the growth in eye anterior chamber and interinduction of apoptosis between tumor cells and splenocytes. These conclusions are confirmed by the data of Narayan and co-authors [Narayan et al. 2000] about hypersensitivity to radiation-induced non-apoptotic and apoptotic death in cell lines from patients with the chromosome instability syndrome. Lymphoblastic cell lines from these patients were characterized by the long-term cell cycle arrest, an increase of apoptosis, and a decrease of clonogenity after irradiation, as compared with normal tissues. Study of the Hodgkin-Reed-Sternberg cell line L1236, with the use of cytogenetic analysis, did not reveal in these cells any specific chromosomal aberrations, whereas use of panels of microsatellite loci allowed to detect regions with loss of heterozygocity in sites of chromosomes 6p, 9q, and 17p [Staratschek-jox et al. 2001].

1.3.4. Conclusion	
Of especial importance is the ability of tumor cells to suppress components of immune system, particularly their ability to affect lymphocyte apoptosis [Bennett et al. 1998; O`Connell et al. 1999; Rabinowich et al. 1998; Reichert et al. 1998]. We showed that the tumor hepatocyte population could be heterogeneous in abilities to induce apoptosis in splenocytes and to undergo splenocyte-induced apoptosis [Ginkul et al. 2001; Shvemberger and Gincul 2002]. We have found that high DNA-polymorphism in hepatocytes revealed by RAPD-PCR correlates with capability of tumor hepatocytes for differentiation at growth in eye anterior chamber. We suggest that genetic rearrangements in the heterogeneous tumor cell population may lead to the appearance of the clones that, on one hand, are more stable to the lymphocyte-induced apoptosis, while, on the other hand, become more aggressive to lymphocytes.

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