Effect of ras-Gene Transformation on the Inhibition of NIH3T3 Cell Growth by Pertussis Toxin
John D. Hildebrandt, Lynne Lederman, and David L. Steffen Worcester Foundation for Experimental Biology (J.D.H., L.L.) Shrewsbury, Massachusetts 01545
To investigate the relationship between the effects of a pertussis toxin-inhibitable class of G-proteins and the ras family of protooncogenes on cell growth, we isolated multiple cell lines transformed by oncogenic Hras or Nras genes and measured the ability of pertussis toxin to inhibit their growth rate. Although all of the cell lines were morphologically transformed and could grow in agar suspension, there was considerable variability in their resistance to pertussis toxin, ranging from cell lines completely resistant to pertussis toxin to cell lines as sensitive to pertussis toxin as the parental cells from which they derived. For those lines resistant to pertussis toxin, this resistance is not due to an inability of pertussis toxin to reach or react with its intracellular target; pertussis toxin could be shown to ADP-ribosylate the endogenous G-proteins of all lines tested regardless of whether it affected their growth rate. There was a strong correlation between the level of active ras protein expressed in the different lines and the degree of resistance to pertussis toxin (r = 0.80). Although the Hras-transformed cell lines were more resistant to pertussis toxin as a group than the Nras-transformed cell lines, we believe that this is not a primary difference between Nras and Hras, but, rather, is due to a higher average level of expression of ras in the cell lines receiving Hras. We suggest that the consequences of ras transformation vary with the concentration of oncogenic ras present in the cell, and that different assays or different properties of transformation show different sensitivities to the level of ras expression. These results have important consequences for the interpretation of experiments that attempt to understand the consequences of transformation by ras oncogenes and the function of the ras gene product. (Molecular Endocrinology 5:1101-1108,1991)
of these genes have been found in a wide variety of different kinds of tumors (1-4). Secondly, this family has been found to be the most evolutionary conserved of all the protooncogenes (5). Thirdly, evidence has been presented that many effectors of normal and abnormal growth control are mediated through ras protooncogenes (6, 7). One of the major unanswered questions about this family of genes is how they regulate cell growth. We and others have shown that the growth rate of a number of different cell lines is inhibited by treatment with pertussis toxin (8-14). This effect appears to be the result of the toxin's ability to inactivate pertussis toxin-sensitive G-protein(s), suggesting a role for such G-proteins in the regulation of cell growth. Given the central role that the ras family of gene products has in the transmission of many growth regulatory signals, we wished to test whether this was true for the pertussis toxin-sensitive signal(s) as well. To this end, we introduced transforming mutant ras oncogenes into NIH3T3 cells and measured the ability of pertussis toxin to alter the growth rate of these transformed cells. We found that although expression of high levels of transforming ras protein is capable of preventing the effect of pertussis toxin, expression of lower (albeit transforming) levels of ras is not. We describe how this dependence on oncogene concentration complicates the interpretation of a number of published results and propose a model for the relationship between pertussis-toxin-sensitive G-proteins and ras to explain this effect.
RESULTS Sensitivity of the Growth of Different Cell Lines to Pertussis Toxin
INTRODUCTION The ras family of protooncogenes is of particular interest for several reasons. Firstly, mutant oncogenic forms
Table 1 lists the cell lines produced for this study. Cell lines containing one of two different ras oncogenes (Hras and Nras) were generated. A number of independent cell lines containing each oncogene were gen-
0888-8809/91/1101-1108$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
1101
Downloaded from https://academic.oup.com/mend/article-abstract/5/8/1101/2714445 by guest on 15 January 2019
Department of Cell Biology Baylor College of Medicine (D.L.S.) Houston, Texas 77030
Vol 5 No. 8
MOL ENDO-1991 1102
Table 1. Cell Lines Generated for This Study Vector
How Introduced
How Cloned
Oncogene
Hras Hras Hras Hras Hras Hras Hras Hras Hras Hras Hras Hras
LJras LJras LJras LJras LJras LJras LJras LJras LJras LJras LJras LJras
Infection Infection Infection Infection Infection Infection Infection Infection Infection Infection Infection Infection
Colony Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter
NIH-Nras1 NIH-Nras4 NIH-Nras9
Nras Nras Nras
pNRsac + pSV2neo pNRsac + pSV2neo pNRsac + pSV2neo
Transfection Transfection Transfection
Colony Colony Colony
NIH-neo4 NIH-r/eo5 NIH-neo8
None None None
pSV2neo pSV2neo pSV2neo
Transfection Transfection Transfection
Colony Colony Colony
erated in order to determine whether clone to clone variations would affect the results of this study. Since the cell lines were isolated by selection for the neo gene, control lines containing pSV2neo alone were also generated and used as negative controls. Figure 1 summarizes the results of nine experiments testing the pertussis toxin sensitivity of the growth rates of the cell lines listed in Table 1. The data are plotted as the percent inhibition of cell growth by pertussis toxin. Six of the 12 Hras-transformed cell lines (50%) exhibited no significant sensitivity to pertussis toxin. The remaining six retained sensitivity to pertussis toxin, but in all six, this sensitivity was significantly less than that of the control NIH3T3 or neo-transfected cell lines. Of the three Nras-transformed lines, all were significantly sensitive to pertussis toxin. While one of the Nras-transformed cell lines was significantly less sensitive than the controls, the other two were not significantly different from the controls.
60 -
50 -
40 -
20 •
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10
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-T-
n J C T J n j n j n j r o c o n J n j c o t / )
i x x i i i x x i r S
Growth of Selected Cell Lines in Agar Suspension All of the ras-containing cell lines were indirectly selected based on a cointroduced neo marker. However, all of them appeared to be transformed by morphology. Because some of them retained pertussis toxin sensitivity, in particular the Nras-transformed lines, it was important to have an objective measure of transformation. Thus, the ability of selected cell lines to grow in agar suspension was tested. For negative controls, the parental NIH3T3 cells and three neo-alone cell lines were also included. The results are summarized in Table 2. All of the ras-transformed cell lines and none of the control lines grew in agar suspension. Interestingly, however, the agar cloning efficiency of different clones varied substantially.
Fig. 1. Effect of Pertussis Toxin on Growth of Normal and Transformed Cells The graph summarizes the results of nine experiments measuring the effect of pertussis toxin on growth rates, as described in Materials and Methods. Values plotted are the mean ± SEM for the percent inhibition of growth by pertussis toxin. The vertical lines separate the control, Hras-transformed, and Nras-transformed cell lines. The solid horizontal line indicates no inhibition, while the dashed horizontal lines indicate the inhibition of growth observed with NIH3T3 cells (1 SD). For each experiment, growth rates were measured in triplicate. The number of experiments for each cell line are: NIH3T3, 8; r/ec-4, 4; neo5, 3; r>eo8, 3; Hrasi, 7; HrasAI, 3; HrasA2, 3; HrasA3, 3; HrasA4, 3; HrasBI, 2; HrasB2, 2; HrasB4, 2; HrasB5, 2; HrasB7, 2; HrasB9, 4; HrasB11, 2; Nrasi, 2; Nras4, 3; Nras9, 2.
Downloaded from https://academic.oup.com/mend/article-abstract/5/8/1101/2714445 by guest on 15 January 2019
Cell Line
NIH-Hras1 NIH-HrasA1 NIH-HrasA2 NIH-HrasA3 NIH-HrasA4 NIH-HrasB1 NIH-HrasB2 NIH-HrasB4 NIH-HrasB5 NIH-HrasB7 NIH-HrasB9 NIH-HrasB11
ras and Pertussis Toxin Effects 1105
ras and Pertussis Toxin Effects
1103
Table 2. Growth of Selected Cell Lines in Soft Agar % CFU
H/-as1 HrasB9 Nrasi Nras4 Nras9 neo4 neo5 neo8 NIH3T3
2 0.4 1 0.8 0.3
John D. Hildebrandt, Lynne Lederman, and David L. Steffen Worcester Foundation for Experimental Biology (J.D.H., L.L.) Shrewsbury, Massachusetts 01545
To investigate the relationship between the effects of a pertussis toxin-inhibitable class of G-proteins and the ras family of protooncogenes on cell growth, we isolated multiple cell lines transformed by oncogenic Hras or Nras genes and measured the ability of pertussis toxin to inhibit their growth rate. Although all of the cell lines were morphologically transformed and could grow in agar suspension, there was considerable variability in their resistance to pertussis toxin, ranging from cell lines completely resistant to pertussis toxin to cell lines as sensitive to pertussis toxin as the parental cells from which they derived. For those lines resistant to pertussis toxin, this resistance is not due to an inability of pertussis toxin to reach or react with its intracellular target; pertussis toxin could be shown to ADP-ribosylate the endogenous G-proteins of all lines tested regardless of whether it affected their growth rate. There was a strong correlation between the level of active ras protein expressed in the different lines and the degree of resistance to pertussis toxin (r = 0.80). Although the Hras-transformed cell lines were more resistant to pertussis toxin as a group than the Nras-transformed cell lines, we believe that this is not a primary difference between Nras and Hras, but, rather, is due to a higher average level of expression of ras in the cell lines receiving Hras. We suggest that the consequences of ras transformation vary with the concentration of oncogenic ras present in the cell, and that different assays or different properties of transformation show different sensitivities to the level of ras expression. These results have important consequences for the interpretation of experiments that attempt to understand the consequences of transformation by ras oncogenes and the function of the ras gene product. (Molecular Endocrinology 5:1101-1108,1991)
of these genes have been found in a wide variety of different kinds of tumors (1-4). Secondly, this family has been found to be the most evolutionary conserved of all the protooncogenes (5). Thirdly, evidence has been presented that many effectors of normal and abnormal growth control are mediated through ras protooncogenes (6, 7). One of the major unanswered questions about this family of genes is how they regulate cell growth. We and others have shown that the growth rate of a number of different cell lines is inhibited by treatment with pertussis toxin (8-14). This effect appears to be the result of the toxin's ability to inactivate pertussis toxin-sensitive G-protein(s), suggesting a role for such G-proteins in the regulation of cell growth. Given the central role that the ras family of gene products has in the transmission of many growth regulatory signals, we wished to test whether this was true for the pertussis toxin-sensitive signal(s) as well. To this end, we introduced transforming mutant ras oncogenes into NIH3T3 cells and measured the ability of pertussis toxin to alter the growth rate of these transformed cells. We found that although expression of high levels of transforming ras protein is capable of preventing the effect of pertussis toxin, expression of lower (albeit transforming) levels of ras is not. We describe how this dependence on oncogene concentration complicates the interpretation of a number of published results and propose a model for the relationship between pertussis-toxin-sensitive G-proteins and ras to explain this effect.
RESULTS Sensitivity of the Growth of Different Cell Lines to Pertussis Toxin
INTRODUCTION The ras family of protooncogenes is of particular interest for several reasons. Firstly, mutant oncogenic forms
Table 1 lists the cell lines produced for this study. Cell lines containing one of two different ras oncogenes (Hras and Nras) were generated. A number of independent cell lines containing each oncogene were gen-
0888-8809/91/1101-1108$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
1101
Downloaded from https://academic.oup.com/mend/article-abstract/5/8/1101/2714445 by guest on 15 January 2019
Department of Cell Biology Baylor College of Medicine (D.L.S.) Houston, Texas 77030
Vol 5 No. 8
MOL ENDO-1991 1102
Table 1. Cell Lines Generated for This Study Vector
How Introduced
How Cloned
Oncogene
Hras Hras Hras Hras Hras Hras Hras Hras Hras Hras Hras Hras
LJras LJras LJras LJras LJras LJras LJras LJras LJras LJras LJras LJras
Infection Infection Infection Infection Infection Infection Infection Infection Infection Infection Infection Infection
Colony Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter Microtiter
NIH-Nras1 NIH-Nras4 NIH-Nras9
Nras Nras Nras
pNRsac + pSV2neo pNRsac + pSV2neo pNRsac + pSV2neo
Transfection Transfection Transfection
Colony Colony Colony
NIH-neo4 NIH-r/eo5 NIH-neo8
None None None
pSV2neo pSV2neo pSV2neo
Transfection Transfection Transfection
Colony Colony Colony
erated in order to determine whether clone to clone variations would affect the results of this study. Since the cell lines were isolated by selection for the neo gene, control lines containing pSV2neo alone were also generated and used as negative controls. Figure 1 summarizes the results of nine experiments testing the pertussis toxin sensitivity of the growth rates of the cell lines listed in Table 1. The data are plotted as the percent inhibition of cell growth by pertussis toxin. Six of the 12 Hras-transformed cell lines (50%) exhibited no significant sensitivity to pertussis toxin. The remaining six retained sensitivity to pertussis toxin, but in all six, this sensitivity was significantly less than that of the control NIH3T3 or neo-transfected cell lines. Of the three Nras-transformed lines, all were significantly sensitive to pertussis toxin. While one of the Nras-transformed cell lines was significantly less sensitive than the controls, the other two were not significantly different from the controls.
60 -
50 -
40 -
20 •
c
10
-10
•20 « < < < < m c o m c o c Q C Q » G j t f ) U i i n U i W t n i f i i f i i f i t f i { T }
-T-
n J C T J n j n j n j r o c o n J n j c o t / )
i x x i i i x x i r S
Growth of Selected Cell Lines in Agar Suspension All of the ras-containing cell lines were indirectly selected based on a cointroduced neo marker. However, all of them appeared to be transformed by morphology. Because some of them retained pertussis toxin sensitivity, in particular the Nras-transformed lines, it was important to have an objective measure of transformation. Thus, the ability of selected cell lines to grow in agar suspension was tested. For negative controls, the parental NIH3T3 cells and three neo-alone cell lines were also included. The results are summarized in Table 2. All of the ras-transformed cell lines and none of the control lines grew in agar suspension. Interestingly, however, the agar cloning efficiency of different clones varied substantially.
Fig. 1. Effect of Pertussis Toxin on Growth of Normal and Transformed Cells The graph summarizes the results of nine experiments measuring the effect of pertussis toxin on growth rates, as described in Materials and Methods. Values plotted are the mean ± SEM for the percent inhibition of growth by pertussis toxin. The vertical lines separate the control, Hras-transformed, and Nras-transformed cell lines. The solid horizontal line indicates no inhibition, while the dashed horizontal lines indicate the inhibition of growth observed with NIH3T3 cells (1 SD). For each experiment, growth rates were measured in triplicate. The number of experiments for each cell line are: NIH3T3, 8; r/ec-4, 4; neo5, 3; r>eo8, 3; Hrasi, 7; HrasAI, 3; HrasA2, 3; HrasA3, 3; HrasA4, 3; HrasBI, 2; HrasB2, 2; HrasB4, 2; HrasB5, 2; HrasB7, 2; HrasB9, 4; HrasB11, 2; Nrasi, 2; Nras4, 3; Nras9, 2.
Downloaded from https://academic.oup.com/mend/article-abstract/5/8/1101/2714445 by guest on 15 January 2019
Cell Line
NIH-Hras1 NIH-HrasA1 NIH-HrasA2 NIH-HrasA3 NIH-HrasA4 NIH-HrasB1 NIH-HrasB2 NIH-HrasB4 NIH-HrasB5 NIH-HrasB7 NIH-HrasB9 NIH-HrasB11
ras and Pertussis Toxin Effects 1105
ras and Pertussis Toxin Effects
1103
Table 2. Growth of Selected Cell Lines in Soft Agar % CFU
H/-as1 HrasB9 Nrasi Nras4 Nras9 neo4 neo5 neo8 NIH3T3
2 0.4 1 0.8 0.3
