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Cell Biology International (2005) 29, 1047–1049 (Printed in Great Britain)
Possible role of endogenous growth inhibitors in regeneration of organs: Searching for new approaches
N. Giorgobiania*, D. Dzidziguria, M. Rukhadzeb, L. Rusishvilia and G. Tumanishvilia
aLaboratory of Developmental Biology, Tbilisi State University, 1 Chavchavadze Avenue, 0108 Tbilisi, Georgia, USA
bLaboratory of Neurobiology, Tbilisi State University, Tbilisi, Georgia, USA


Abstract

A hydrophilic protein component (12–17kDa) of the thermostable protein complex (TSPC) of different organs (heart, liver, kidney, brain) of adult white rats has been identified using hydrophobic interaction chromatography (HIC). Chromatograms and spectra of the myocardial hydrophilic component of different animals (snail, pigeon, rat, pig) detected within the UV region (190–360nm) have shown that in different organs of the same animal species as well as in the same organ (e.g. heart) of different species, TSPC contains identical hydrophilic components, i.e. there is clearly a phylogenetically conserved group of thermostable proteins that regulates proliferation processes.


Keywords: Endogenic growth inhibitor, Growth regulation.

*Corresponding author.


1 Introduction

Endogenous regulators of proliferation are protein molecules, encoded by regulating genes. They send specific signals to target cells to induce proliferation. In our opinion, one of the topical trends in the field of regulation of cell proliferation is investigation of endogenous growth inhibitory factors. Growth regulatory protein factors have been obtained from different organs of adult animals (Balazs and Blazsek, 1979; Kusen and Stoika, 1985; Amano and Iseki, 2001); at those times, comparatively scanty data existed about growth inhibitory factors of the myocardium. Most investigations included non-specific factors that affect: (a) the expression of synthesis of the different myocardium proteins; (b) the hypertrophy of cardiomyocytes; and (c) a directed action against death of cardiomyocytes after infarction (Kardami, 1990; Detillieux et al., 2003; Nishida et al., 2003; Kardami et al., 2004).

In general, our interest is mainly in specific growth inhibitory factors of myocardium and identification of similar factors in other organs. The objective of the present work was growth inhibitory factors obtained from: (i) the myocardium of animals of different phylogenetic stages; and (ii) different organs of the same animal.

2 Materials and methods

Myocardium, liver, kidney, and brain of white adult rats, as well as myocardium of adult snail, pigeon, and pig, were used as a material for investigation. Thermostable protein fractions were obtained by the method of alcohol precipitation of Balazs and Blazsek (1979), with modification. Animals were decapitated under diethyl ether. Organs were removed quickly, separated from capsules of connective tissues and vessels, rinsed with the physiological solution, and crushed. Aqueous homogenates were prepared in a tissue/distilled water ratio of 1:8. The homogenates were saturated step-wise with 96% ethanol to obtain 81% ethanol fraction, which was heated in a water bath (100°C) for 20min, cooled and centrifuged (600×g, 15min). The supernatant was frozen in liquid nitrogen and dried in an absorptive-condensate lyophilizer. As a result a residue was obtained of a thermostable protein complex (TSPC), a light-gray powder soluble in water. Samples were kept at 4°C. Protein concentration was determined by the method of Lowry et al. (1951).

Hydrophobic interaction chromatography (HIC) was used for comparative analysis of TSPC (Queiroz et al., 2001). A hydrophilic polymeric sorbent, HEMA BIO Phenyl-1000 (particle size 10μm) modified by phenyl groups, served as the stable phase. The mobile phase was phosphate buffer (pH 7.4) with ammonium sulfate. Elution was performed with the mobile phase in molar concentration range from 2.0M to 0.0M (pure buffer) with respect to (NH4)2SO4. For co-elution of hydrophilic and hydrophobic components of protein fractions, Brij-35 polyoxyethylene dodecyl ether with increasing concentration from 0% to 3% was added to the mobile phase. UV detection was usually set at 230nm.

3 Results and discussion

The chromatograms in Fig. 1 show that TSPC extracted from the hearts of the animals (snail, pigeon, rat, pig) contains a hydrophilic component with a retention time of 5.5min (corresponding to the retention time of cytochrome c). Elevation of the baseline was occurred after 30min, indicating the existence of a hydrophobic component in the protein fraction of TSPC. Elution of the hydrophobic component with a reasonable retention time of 15min was attained by the addition of an increasing quantity of Brij-35 in the mobile phase (Fig. 2).


Fig. 1

Chromatograms of TSPC extracted from the heart of snail, pigeon, pig and rat.


Fig. 2

The hydrophilic (1) and hydrophobic (2) components of TSPC.



In previous work, we found that TSPC extracted from heart, liver, kidney, and brain of adult white rats inhibits both proliferation and transcription on average by 40–50% in cells of homologous organs of 7-day-old rats (Salakaya et al., 2000; Giorgobiani et al., 2002, 2003; Rusishvili et al., 2003; Rukhadze et al., 2005). We had also determined the molecular weights of TSPC components. In particular, electrophoresis in polyacrylamide gel showed the presence of a low molecular weight subfraction of 12–17kDa and a relatively high molecular weight component of 60–70kDa.

Focusing our attention here on the hydrophilic component of TSPC, the problem is whether this hydrophilic component and the low molecular weight fraction described earlier (Giorgobiani et al., 2002) are the same fraction. Chromatography of the low molecular weight TSPC component (Fig. 3) shows that the 12–17kDa subfraction (similar to cytochrome c) with mitosis-inhibiting ability is the hydrophilic component of TSPC. Fig. 4 gives the spectrum of the hydrophilic component in the UV region from 190 to 360nm).


Fig. 3

Chromatogram of low molecular weight TSPC component.


Fig. 4

Spectrum of hydrophilic component in the UV region (190–360nm).



Chromatograms in Fig. 5 show hydrophilic and hydrophobic components of TSPC extracted from different organs (heart, liver, kidney, brain) of the same animal (adult white rat). According to the results, hydrophilic fractions of TSPC of all investigated organs seemed identical. The presence of the hydrophilic fraction in the myocardium of different animals indicates that a phylogenetically conservative group of thermostable proteins inhibiting proliferation processes has been found.


Fig. 5

Chromatograms of hydrophilic and hydrophobic components of TSPC extracted from different organs (heart, liver, kidney, brain) of rat.


In the light of these results, we suggest two approaches for the use of hydrophilic (12–17kDa) fraction of TSPC in organ growth regulation: first, production of antibodies to the proteins of TSPC hydrophilic component and their use for stimulation of proliferation processes in organs with a limited potential to regenerate, such as myocardium; and second, we would propose the use of TSPC hydrophilic fraction as an inhibitor in organ growth regulation during the postnatal period.

References

Amano O, Iseki, S. Expression and localization of cell growth factors in the salivary gland. Kaibogaku Zasshi 2001:76:1, 2:201-12
Medline   1st Citation  

Balazs A, Blazsek, I. Control of cell proliferation by endogenous inhibitors. Akademia Kiado (Budapest) 1979:302:
1st Citation   2nd  

Detillieux KA, Sheikh, F, Kardami, E, Cattini, PA. Biological activities of fibroblast factor-2 in the adult myocardium. Cardiovasc Res 2003:57:8-19
Crossref   Medline   1st Citation  

Giorgobiani N, Chkhobadze, M, Rusishvili, L, Dzidziguri, D, Tumanishvili, G. Study of endogenic growth inhibitors in different organs of white rats. Proc Georgian Acad Sci Ser B 2003:9:46-9
1st Citation  

Giorgobiani N, Rusishvili, L, Dzidziguri, D, Tumanishvili, G. The study of nucleolar parameter in evaluation of the rat myocardium functional state after the treatment with specific growth-inhibitory cardiomyocyte factor. Proc Georgian Acad Sci 2002:28:Suppl:27-31
1st Citation   2nd  

Kardami E. Stimulation and inhibition of cardiac myocyte proliferation in vitro. Mol Cell Biochem 1990:92:129-35
Medline   1st Citation  

Kardami E, Jiang, ZS, Jimenes, SK, Hirst, CJ, Sheikh, F, Zahradka, P. Fibroblast growth factor 2 isoforms end cardiac hypertrophy. Cardiovasc Res 2004:63:3:458-66
Crossref   Medline   1st Citation  

Kusen SI, Stoika, PS. Molecular mechanisms in action of polypeptide growth-factors. 1985:
1st Citation  

Lowry DH, Rosebrough, NJ, Farr, AL, Randell, RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951:193:265-75
Medline   1st Citation  

Nishida S, Nagamine, H, Tanaka, Y, Watanabe, G. Protective effect of basic fibroblast growth factor against myocyte death and arrhythmias in acute myocardial infarction in rats. Circ J 2003:67:334-9
Crossref   Medline   1st Citation  

Queiroz JA, Tomaz, CT, Cabral, JMS. Hydrophobic interaction chromatography of proteins. J Chromatogr 2001:87:143-59
1st Citation  

Rukhadze M, Dzidziguri, D, Giorgobiani, N, Kerkenjia, S. The study of growth inhibitive protein factor by various mode of HPLC and estimation of its binding with drugs. Biomed Chromatogr 2005:19:36-42
Crossref   Medline   1st Citation  

Rusishvili L, Giorgobiani, N, Dzidziguri, D, Tumanishvili, G. Comparative analysis of cardiomyocyte growth inhibitive factor in animals of different classes. Proc Georgian Acad Sci Ser B 2003:1:1–2:42-5
1st Citation  

Salakaya T, Giorgobiani, N, Dzidziguri, D, Tumanisvili, G. Further study of the growth inhibiting factor (GIF) isolated from rat ventricular myocardium. Bull Georgian Acad Sci 2000:162:175-7
1st Citation  


doi:10.1016/j.cellbi.2005.10.012


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)