394
Biochimica et Biophysica Acta, 5 8 3 ( 1 9 7 9 ) 3 9 4 - - 4 0 2 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28819
INHIBITION OF CELL-SUBSTRATUM ATTACHMENT OF CULTURED RAT HEART CELLS BY PROTEIN SYNTHESIS INHIBITORS
D A V I D W. S P E I C H E R * a n d R I C H A R D L. M c C A R L **
Department of Biochemistry and Biophysics, The Pennsylvania State University, University Park, PA 16802 (U.S,A.) (Received May 17th, 1978) ( R e v i s e d m a n u s c r i p t received O c t o b e r 1 0 t h , 1 9 7 8 )
Key words: Protein synthesis inhibitor; Cell substratum attachment; mRNA formation; Adhesion; (Rat heart cell)
Summary Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were most dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachment completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.
* Present address: Department of Pathology, Yale Medical School, 310 Cedax Street, New Haven, CT 06511. ** To whom correspondence should be addressed.
395 Introduction During early studies of cell dissociation by proteolytic enzymes it was believed that proteolytic enzymes, especially crude trypsin did n o t damage living cells [1]. However, electron microscopy of trypsin-dissociated cells showed severe external and internal membrane damage [2] necessitating a reevaluation of previous concepts. Further work showed that trypsin and other proteases were adsorbed to cell surfaces and remained in an active form for as long as 24 h after enzyme treatment [3--6]. Recently, investigators found that trypsin penetrated cells and could apparently damage internal structures in addition to the cell membrane [7--9]. Mild treatment of erythrocyte membranes with trypsin and other proteases resulted in changes in morphology and release of glycopeptides [ 1 0 - - 1 3 ] . Proteolytic enzymes also hydrolyzed glycoproteins in platelet membranes [14] and protease treatment of adipose tissue digested the insulin receptor [15]. Loss of membrane glycoproteins and high molecular weight polypeptides believed to play a role in membrane stabilization and cell shape have been observed [16]. Membrane protein loss also has been observed in chick e m b r y o fibroblasts as a result of enzyme treatment [17]. Membrane protein alterations in response to trypsin treatment also have been implicated in altered HeLa cell colony morphology [18] and in appearance of malignant cell antigens in normal cells [19]. Glycoproteins on the cell surface probably are involved in cell-substratum adhesion as is evidenced by two mutants of Balb/c 3T3 cells which are defective in adhesiveness for the substratum. Lactoperoxidase-catalyzed iodination of the cell surface showed the absence of two major polypeptides in the mutant and a decrease in the concentration of a third polypeptide [20]. When the major cell surface glycoprotein of chick e m b r y o flbroblasts is added to a variety o f transformed cells, the transformed cells show increased adhesion to the substratum [21]. Loss of a low molecular weight cell surface protein o f chick e m b r y o fibroblasts as a result of protease treatment has also been correlated with decreased cellular attachment and flattening [17]. When attached cells are removed from a substratum they leave behind glycoproteins firmly attached to the substratum. This occurs even when the cells are removed using divalent cation chelators [22,23]. This 'substrate-attached material' is apparently involved in attachment of cells to the substratum. If cells are added to surfaces containing 'substrate-attached material', attachment progresses faster and adhesion is stronger. In both the presence and absence of 'substrate-attached material', trypsin-treated cells attach more slowly than EDTAdissociated cells. This apparently reflects the more extensive cell surface damage caused by the trypsin. A longer recovery period is required in order to repair this damage. When transformed cells are plated on 'substrate-attached material' containing substrates, attachment is increased and cellular morphology resembles that of non-transformed cells [24]. Treatment of cells with proteases could reasonably be expected to result in damage to proteins involved in adhesion with a resulting altered and/or delayed attachment. Evidence indicates that this is indeed the case. Protease treatment may result also in extensive membrane damage which requires at least several hours to repair. In addition, the possibility exists that either new proteins or
396 higher concentrations of existing proteins are required for cell-substratum binding. If one or both of these events are required for attachment then protein synthesis should be required for cell-substrate attachment. To test the hypothesis that protein synthesis is required for cell-substrate attachment of neonatal rat heart cells, the effects of cycloheximide and actinomycin D on attachment were investigated. Cycloheximide has been used as an inhibitor of peptide chain elongation in eukaryotic organisms [ 25]. Actinomycin D intercalates into DNA regions rich in guanine-cytosine pairs to prevent transcription [26]. Thus actinomycin D affects protein synthesis at the level of transcription. Proteins synthesized from short-lived mRNAs are inhibited first by actinomycin D, while protein synthesis utilizing long-lived m R N A is not affected until later times. Experimental procedure
Heart cell culture procedure. Hearts of two to five
Biochimica et Biophysica Acta, 5 8 3 ( 1 9 7 9 ) 3 9 4 - - 4 0 2 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28819
INHIBITION OF CELL-SUBSTRATUM ATTACHMENT OF CULTURED RAT HEART CELLS BY PROTEIN SYNTHESIS INHIBITORS
D A V I D W. S P E I C H E R * a n d R I C H A R D L. M c C A R L **
Department of Biochemistry and Biophysics, The Pennsylvania State University, University Park, PA 16802 (U.S,A.) (Received May 17th, 1978) ( R e v i s e d m a n u s c r i p t received O c t o b e r 1 0 t h , 1 9 7 8 )
Key words: Protein synthesis inhibitor; Cell substratum attachment; mRNA formation; Adhesion; (Rat heart cell)
Summary Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were most dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachment completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.
* Present address: Department of Pathology, Yale Medical School, 310 Cedax Street, New Haven, CT 06511. ** To whom correspondence should be addressed.
395 Introduction During early studies of cell dissociation by proteolytic enzymes it was believed that proteolytic enzymes, especially crude trypsin did n o t damage living cells [1]. However, electron microscopy of trypsin-dissociated cells showed severe external and internal membrane damage [2] necessitating a reevaluation of previous concepts. Further work showed that trypsin and other proteases were adsorbed to cell surfaces and remained in an active form for as long as 24 h after enzyme treatment [3--6]. Recently, investigators found that trypsin penetrated cells and could apparently damage internal structures in addition to the cell membrane [7--9]. Mild treatment of erythrocyte membranes with trypsin and other proteases resulted in changes in morphology and release of glycopeptides [ 1 0 - - 1 3 ] . Proteolytic enzymes also hydrolyzed glycoproteins in platelet membranes [14] and protease treatment of adipose tissue digested the insulin receptor [15]. Loss of membrane glycoproteins and high molecular weight polypeptides believed to play a role in membrane stabilization and cell shape have been observed [16]. Membrane protein loss also has been observed in chick e m b r y o fibroblasts as a result of enzyme treatment [17]. Membrane protein alterations in response to trypsin treatment also have been implicated in altered HeLa cell colony morphology [18] and in appearance of malignant cell antigens in normal cells [19]. Glycoproteins on the cell surface probably are involved in cell-substratum adhesion as is evidenced by two mutants of Balb/c 3T3 cells which are defective in adhesiveness for the substratum. Lactoperoxidase-catalyzed iodination of the cell surface showed the absence of two major polypeptides in the mutant and a decrease in the concentration of a third polypeptide [20]. When the major cell surface glycoprotein of chick e m b r y o flbroblasts is added to a variety o f transformed cells, the transformed cells show increased adhesion to the substratum [21]. Loss of a low molecular weight cell surface protein o f chick e m b r y o fibroblasts as a result of protease treatment has also been correlated with decreased cellular attachment and flattening [17]. When attached cells are removed from a substratum they leave behind glycoproteins firmly attached to the substratum. This occurs even when the cells are removed using divalent cation chelators [22,23]. This 'substrate-attached material' is apparently involved in attachment of cells to the substratum. If cells are added to surfaces containing 'substrate-attached material', attachment progresses faster and adhesion is stronger. In both the presence and absence of 'substrate-attached material', trypsin-treated cells attach more slowly than EDTAdissociated cells. This apparently reflects the more extensive cell surface damage caused by the trypsin. A longer recovery period is required in order to repair this damage. When transformed cells are plated on 'substrate-attached material' containing substrates, attachment is increased and cellular morphology resembles that of non-transformed cells [24]. Treatment of cells with proteases could reasonably be expected to result in damage to proteins involved in adhesion with a resulting altered and/or delayed attachment. Evidence indicates that this is indeed the case. Protease treatment may result also in extensive membrane damage which requires at least several hours to repair. In addition, the possibility exists that either new proteins or
396 higher concentrations of existing proteins are required for cell-substratum binding. If one or both of these events are required for attachment then protein synthesis should be required for cell-substrate attachment. To test the hypothesis that protein synthesis is required for cell-substrate attachment of neonatal rat heart cells, the effects of cycloheximide and actinomycin D on attachment were investigated. Cycloheximide has been used as an inhibitor of peptide chain elongation in eukaryotic organisms [ 25]. Actinomycin D intercalates into DNA regions rich in guanine-cytosine pairs to prevent transcription [26]. Thus actinomycin D affects protein synthesis at the level of transcription. Proteins synthesized from short-lived mRNAs are inhibited first by actinomycin D, while protein synthesis utilizing long-lived m R N A is not affected until later times. Experimental procedure
Heart cell culture procedure. Hearts of two to five
