Printed in Sweden Copyright Q 1975 by Academic Press, Inc. All rights o.f reproduction in any form reserved
Experimental Cell Research91 (1975) 422-428
RELATIONSHIP
BETWEEN CYCLIC AMP, MICROTUBULE
ORGANIZATION,
AND MAMMALIAN
CELL SHAPE
Studies on Chinese Hamster Ovary Cells and their Variants LINDA
S. BORMAN,l
J. N. DUMONT,Z and A. W. HSIE2
IThe University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and the 2Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830, USA
SUMMARY Treatment of Chinese hamster ovary cells with N6,2’-O-dibutyryl adenosine cyclic 3’,5’-monophosphate (db-CAMP) and hormones converts their shape from a knobbed, epithelial-like morphology to. a smooth fibroblast-like form. Ultrastructural studies demonstrate an increased number of microtubules and their arrangement in parallel array along the long axis of the cell after treatment with these agents. Although an epithelial-like variant, when treated with dbCAMP, shows an increase in the number of microtubules; these microtubules remain in a disorganized nonparallel array. The numerous long microtubules which are already present in a fibroblast-like variant become further elongated when the cells are treated with db-CAMP. These experiments establish the relationship between CAMP level, assembly and organization of microtubules, and cell shape in cultured Chinese hamster cells.
It has been demonstrated that treatment of Chinese hamster ovary cell clone K, (CHOK, cells) with N6,2’-O-dibutyryl adenosine 3’,5’-monophosphate (db-CAMP) cyclic alone, or in combination with hormones such as testosterone and prostaglandin El, converts their shape from an epithelial cell-like morphology to a fibroblast-like form [l-3]. This morphological conversion is accompanied by the appearance of characteristics common to the fibroblastic state [24]. Agents like Colcemid and vinblastine, which inhibit assembly of microtubules, prevent conversion from the epithelial-like to the fibroblastlike form. Therefore, it has been postulated that the morphological transformation agents act by promoting the assembly of microtubules from protein monomers [2]. It has also been shown that one of the first manifestaExptl Cell Res 91 (1975)
tions of the action of morphological transformation agents is to cause the disappearance of knob-like protrusions from the periphery of the epithelial-like cells [2, 31. These rounded knobs, which are supported by a small stalk of cytoplasm [5, 61 appear to be centers of active motion of the cell membrane [3]. In the course of studies on the molecular mechanisms associated with morphological transformation, variants have been cloned which differ from the parental CHO-K, cells in their response to db-CAMP with or without testosterone after standard mutagenesis treatment with ethyl methanesulfonate. The morphological characteristics of such variants are reported here. One variant, M-7, displays a constant epithelial-like morphology and is refractory to these hormonal
CAMP, microtubules, and cell morphology
agents. However, when treated with these agents, the number of microtubules appears to increase, although they do not become oriented into parallel arrays. Another clone, designated M-6, assumes a constant fibroblast-like appearance; it has a smooth membrane and contains many parallel arrays of microtubules, even when grown in basal medium.
423
lead citrate, and examined with an Hitachi EMU-11 electron microscope operated at 75 KV. Cells grown on coverslips for RNA staining were fixed with 10 % formalin for 4 min, washed and stained with 1% toluidine blue for 10 min at 37°C rinsed, rapidly dehydrated, and mounted on slides with Permount. Control cells were treated with RNase (Sigma Chemical Co.) for 1 h at 37°C prior to toluidine blue staining.
RESULTS Parental CHO-K, cells
MATERIALS AND METHODS Cell culture CHO-K, cells were routinely grown in medium F12 supplemented with 10 % fetal-calf serum in an incubator at 37°C. 5 % COz. and 100 % humidity, as previously described [2]. Cells for the experiments presented here were removed from stocks by trypsinization and replated in medium F 12 plus 10 % extensively dialysed serum so as to have a more defined growth environment. Treated cells received 1 mM db-CAMP in the medium for a period of 16-24 h before further treatment for microscopy. Such a treatment caused the intracellular CAMP level to rise from l-2 ,L~Mto approx. 20 ,LLM[7]. As we have demonstrated previously, equal molar concentrations of the unsubstituted CAMP. sodium butvrate. adenosine, adenine, as well as various other cyclic nucleotides are inactive Il. 2. 71. Variants M-6 and M-7 are derived from &I&K,cells after treatment with ethyl methanesulfonate. Some of their properties are described in Results.
Microscopy CHO-K, and morphological variants M-6 and M-7 were grown from stock cultures at 60% confluency by trypsinization and transferred to 35-mm dishes (Falcon) containing carbon-coated coverslips and 4 ml of growth medium. When the cells had attached to the coverslips and grown to the desired density50 % for phase microscopy and 80% for electron microscopy-new medium was added, with or without 1 mM db-CAMP, and the cells were allowed to grow for an additional 16-24 h. The medium was then removed and the cells were washed twice with Puck’s Saline G [8] at 25°C. Cells were fixed for 1 h at 35°C in 3 % glutaraldehyde in 0.05 M cacodylate buffer at pH 7.2. The fixed cells were washed with Saline G and postfixed in 1 56 osmium tetroxide in 0.3 M cacodylate buffer. The cells, still attached to coverslips, were then dehydrated and infiltrated with Epon. The coverslins were quartered and placed, cell-side down, on Eponfilled Beem capsules and polymerized at 60°C. After polymerization the covershps were removed and the cells sectioned with a Porter-Blum MT-2 ultramicrotome. The sections were placed on Formvar- and carbon-coated grids, stained with uranyl acetate and
The existence of the knob-like pseudopodial structures at the periphery of the CHO cells (fig. la) has previously been demonstrated by light [3], transmission and scanning electron microscopy [5], and by time-lapse cinematography [4]. Under conditions of morphological transformation, the knobs disappear and the cells assume an elongated, fibroblast-like form with smooth membrane contours [3, 41 (fig. 2a). Toluidine blue staining demonstrates that these knobs are rich in RNA. Electron microscopic studies reveal that the knobs contain an abundant population of polyribosomes, but no other organelles. A few short, randomly oriented microtubules are present in the cytoplasm. There is a band of subplasmalemma1 microfilaments present not only in the knobs but around the entire periphery of the cell (fig. 1b). The most salient feature of morphological transformation of CHO-K, cells is their conversion to an elongated fibroblast-like form [l-3]. This conversion is prevented by agents which inhibit microtubular assembly [l, 21. In the presenceof morphological transformation agents, the microtubules elongate, increase in number, and become arrayed parallel to the long axis of the cell [6]. Microfilaments remain beneath the cell membrane (fig. 26). These observations substantiate data from previous inhibitor studies which show that one of the actions of morphological transformation agents is to promote the asExptl Cell Res 91 (1975)
424
Borman, Dumont and Hsie
Fig. 1. (a) A phase-contrast micrograph of the CHO-K, epithelial-like cells. The arrows point to phase-dense knobs at the periphery of the cells. x 1 000. (b) An electron micrograph of a knob for a CHO-K, cell. The cytoplasm of the knob consists largely of ribosomes. A band of microfilaments is present subjacent to the plasmalemma. The arrows indicate short, randomly placed microtubules. x 14 500.
sembly of microtubules and to convert the cell to the fibroblast-like form [l-3]. In the absence of morphological transformation agents, there are but a few short microtubules randomly distributed within the cytoplasm, usually in the centriolar region (see also discussions in refs [4, 51). Similar observations have been made by Porter et al. [6]. Exptl Cell Res 91 (1975)
The epithelial-like
variant, M-7
In the course of studying the induction of specific gene mutations affecting nutritional dependence and temperature sensitivity of CHO-K, cells by various physical and chemical mutagens, several morphological variant clones have been isolated. A variant, M-7, resembling the parental cell CHO-K,
CAMP, microtubules, and cell morphology
425
Fig. 2. (a) A phase-contrast micrograph of the fibroblast-like appearance of CHO-K, cells after treatment with 1 mM db-CAMP for 24 h and that the knobs have disappeared and the surface membrane is smooth. x 800. (b) An electron micrograph of the margin of a db-CAMP-treated CHO-K, cell. A band of microfilaments (MF) is present beneath the membrane. Profiles of long microtubules (MT), oriented parallel to the long axis of the cell, are also presmt. x 45 000.
cell in that it possesses a typical compact epithelial cell-like appearance with knoblike structures around the periphery has also been isolated. This variant cell line retains its epithelial-like form (fig. 3a) in the presence of morphological transformation agents for as long as 6-7 days. Despite the fact that the cells maintain their epithelial-like shape after treatment with db-CAMP, there is an apparent increase in the number and length of randomly arranged microtubules (fig. 3b). It appears as though the morphological transformation agents have promoted the assembly of microtubules in M-7 cells but not their organization into parallel arrays and the concomitant conversion of the cells to a fibroblast-like morphology.
The fibroblast-like
variant, M-6
Another variant, M-6, assumes a fibroblastlike morphology with a smooth membrane and parallel arrays of microtubules, when grown in standard basal medium, similar to the morphological transformation-agent-induced fibroblast-like form of CHO-K, cells (fig. 2a, b). Under conditions of morphological transformation, the M-6 cells become even further elongated (compare figs 2a and 4a). Exaggerated long, very thin, polar extensions which contain ribosomes, microfilaments, and parallel arrays of microtubules are observed (fig. 4b). Although an increase in the numbers of microtubules under these conditions has not been established, there is an apparent increase in the length of individual microtubules. Exptl Cell Res 91 (1975)
426 Borman, Dumont and Hsie
Fig. 3. (a) A phase contrast of a db-CAMP-treated (1 mM) M-7 epitheliallike variant cell. These cells, with or without treatment, are essentially identical with the untreated CHO-KI cells. x 800. (6) An electron micrograph of a portion of a db-CAMP-treated M-7 cell. Note the long-profile microtubules oriented randomly in the cytoplasm. x 14 500.
Exptl Cell Res 91 (1975)
CAMP, microtubules, and cell morphology
421
Fig. 4. (a) A phase-contrast micrograph cf rib-CAMP-treated (1 mM) fibroblast-like M-6 ceils. These cells become more elongated than similarly treated CHO-K, cells. x 800. (b) An electron micrograph of the elongated process of several db-CAMP-treated M-6 variant cells showing the presence of microfilaments and long microtubules in these extended processes. ’ 33 500.
DISCUSSION In mammalian cells CAMP or its derivatives appear to influence the regulation of many biochemical processes related to growth and differentiation [2, 3, 91. One of the effects of db-CAMP appears to be the promotion of microtubule assembly [I, 21. The present ultrastructural study demonstrates that under morphological transformation conditions there is an increase in the number and orientation of microtubules in CHO-K, cells, an observation also established by others ([6], see also discussions in refs [4, 51). In other cases, for example in mouse neuroblastoma cells, exposure to db-CAMP appears to stabilize the microtubules in neurites so that, during exposure to cold, the neurites do not retract into the cell body [lo]. Microtubules are ubiquitous structural
organelles of eukaryotic cells. They are regarded as playing an essential role in the maintenance of cellular morphology. The existence of random arrays of a small number of relatively short microtubules in the epithelial-like CHO-K, and M-7 cells (see figs 1b and 3b) and, on the other hand, of abundant microtubules highly organized along the long axis of either the bonafide fibroblast-like M-6 cells or the morphological transformation-agent-induced fibroblast-like CHO-K, cells (see figs 2b and 4b) provides direct evidence for the role of microtubules in conferring cell form. Although the loss of microtubule integrity has not been established, it is well known that when normal fibroblast-like, contact-inhibited cells are transformed by viruses, X-radiation, or certain chemical carcinogens they are often converted to a more epithelial-like noncontact-inhibited Exptl Cell Res 91 (1975)
428 Borman, Dumont and Hsie form [ll]. Morphologic conversion, however, which does involve the assembly and reorganization of microtubules, is associated with reversal of certain malignant characteristics of CHO cells in vitro [2-41. It thus becomes important to assess the role of microtubules, or agents which affect them, in malignant transformations. Recently, we have demonstrated that the addition of 1 mM db-CAMP to CHO-K1 cells grown under the conditions used in this study (50-80 % confluency) resulted in an increase of the endogenous level of CAMP from l-2 PM to approx. 20 ,uM, and that it remains at that level for 24 h. This increase of intracellular CAMP level appears to be due largely to the inhibitory action of intracellular N6-monobutyryl CAMP on a low Km CAMP phosphodiesterase, which results in a decreased rate of degradation of intracellular CAMP [7]. Treatment of M-6 and M-7 cells with 1 mM db-CAMP resulted in an increase of intracellular CAMP level of similar magnitude in these cells (P. O’Neill & A. Hsie. Unpublished data). The observation that, when M-7 cells are exposed to db-CAMP, the microtubules are present in increased numbers although in random orientation suggests that M-7 variant cells are capable of responding by increased microtubule assembly, but not by organizing the microtubules into ordered parallel arrays to promote fibroblastlike morphology. By the same token, the fibroblast-like M-6 cells also appear to respond by increasing the length of preexisting oriented microtubules. The experiments with M-7 cells also suggest that the assembly and the organization of microtubules are likely to be two distinct biological processes since the assembly does not necessarily lead to a change in cell form. Changes in cell form appear to be more directly dependent upon the orientation of microtubules. Studies presented in this paper suggest the Exptl Cell Res 91 (1975)
role of intracellular CAMP, through yet unknown mechanisms, in promoting microtubule assembly and organization. In view of (i) recent findings on the involvement of calcium in microtubule polymerization in vitro [12, 131 and (ii) the accumulation of evidence on the interrelationship between calcium metabolism and CAMP in many biological systems [14, 151, we have begun studies of a possible relationship between calcium and CAMP in this system. However, a more direct role of CAMP in effecting microtubule assembly in vivo cannot be excluded. The highly competent technical assistance of Patricia A. Brimerand Nina Hammer in the cell culture work, and of Carole S. King in electron microscopy, is gratefully acknowledged. We thank J. S. Cook and T. Yamada for critical review of the manuscript. L. S. B. is a predoctoral fellow of the University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, supported by grant GM 1974 from the National Institute of General Medical Sciences, NIH. Oak Ridge National Laboratory is operated by Union Carbide Corporation for the US AEC.
REFERENCES 1. Hsie. A W & Waldren. C A., J cell biol 47 (1970), 92 A: Abstr. 2. Hsie. A W & Puck. T T. Proc natl acad sci US 68 (1971) 358. ’ 3. Hsie, A W, Jones, C & Puck, T T, Proc natl acad sci US 68 (1971) 1648. 4. Puck, T T, Waldren, C A & Hsie, A W, Proc natl acad sci US 69 (1972) 1943. 5. Porter, K, Prescott, D & Frye, J, J cell biol 57 (1973) 815. 6. Porter, K R, Puck, T T, Hsie, A W & Kelley, D, Cell 2 (1974) 145. 7. Hsie, A W; Kawashima, K, O’NeilI, J P & Schriider, C H. J biol them (1974). In press. 8. Ham, R’G & Puck, T T, Methods enzymol 5 (1962) 90. 9. Kram, P, Mamont, P & Tomkins, G M, Proc natl acad sci US 70 (1973) 1432. 10. Kirkland, W L & Burton, P R, Nature new biol 240 (1972) 205. 11. Sachs, L, Curr top dev biol 2 (1967) 129. 12. Weisenberg, R C, Science 177 (1972) 1104. 13. Borisy, G G & Olmsted, J B, Science 177 (1972) 1196. 14. Rasmussen, H, Goodman, D B & Teenhouse, A, Crit rev biochem 1 (1972) 95. 15. Whitfield, J F, Rixon, R H, MacManus, J P & Balk, S D, In vitro 8 (1973) 257. Received July 4, 1974
Experimental Cell Research91 (1975) 422-428
RELATIONSHIP
BETWEEN CYCLIC AMP, MICROTUBULE
ORGANIZATION,
AND MAMMALIAN
CELL SHAPE
Studies on Chinese Hamster Ovary Cells and their Variants LINDA
S. BORMAN,l
J. N. DUMONT,Z and A. W. HSIE2
IThe University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and the 2Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830, USA
SUMMARY Treatment of Chinese hamster ovary cells with N6,2’-O-dibutyryl adenosine cyclic 3’,5’-monophosphate (db-CAMP) and hormones converts their shape from a knobbed, epithelial-like morphology to. a smooth fibroblast-like form. Ultrastructural studies demonstrate an increased number of microtubules and their arrangement in parallel array along the long axis of the cell after treatment with these agents. Although an epithelial-like variant, when treated with dbCAMP, shows an increase in the number of microtubules; these microtubules remain in a disorganized nonparallel array. The numerous long microtubules which are already present in a fibroblast-like variant become further elongated when the cells are treated with db-CAMP. These experiments establish the relationship between CAMP level, assembly and organization of microtubules, and cell shape in cultured Chinese hamster cells.
It has been demonstrated that treatment of Chinese hamster ovary cell clone K, (CHOK, cells) with N6,2’-O-dibutyryl adenosine 3’,5’-monophosphate (db-CAMP) cyclic alone, or in combination with hormones such as testosterone and prostaglandin El, converts their shape from an epithelial cell-like morphology to a fibroblast-like form [l-3]. This morphological conversion is accompanied by the appearance of characteristics common to the fibroblastic state [24]. Agents like Colcemid and vinblastine, which inhibit assembly of microtubules, prevent conversion from the epithelial-like to the fibroblastlike form. Therefore, it has been postulated that the morphological transformation agents act by promoting the assembly of microtubules from protein monomers [2]. It has also been shown that one of the first manifestaExptl Cell Res 91 (1975)
tions of the action of morphological transformation agents is to cause the disappearance of knob-like protrusions from the periphery of the epithelial-like cells [2, 31. These rounded knobs, which are supported by a small stalk of cytoplasm [5, 61 appear to be centers of active motion of the cell membrane [3]. In the course of studies on the molecular mechanisms associated with morphological transformation, variants have been cloned which differ from the parental CHO-K, cells in their response to db-CAMP with or without testosterone after standard mutagenesis treatment with ethyl methanesulfonate. The morphological characteristics of such variants are reported here. One variant, M-7, displays a constant epithelial-like morphology and is refractory to these hormonal
CAMP, microtubules, and cell morphology
agents. However, when treated with these agents, the number of microtubules appears to increase, although they do not become oriented into parallel arrays. Another clone, designated M-6, assumes a constant fibroblast-like appearance; it has a smooth membrane and contains many parallel arrays of microtubules, even when grown in basal medium.
423
lead citrate, and examined with an Hitachi EMU-11 electron microscope operated at 75 KV. Cells grown on coverslips for RNA staining were fixed with 10 % formalin for 4 min, washed and stained with 1% toluidine blue for 10 min at 37°C rinsed, rapidly dehydrated, and mounted on slides with Permount. Control cells were treated with RNase (Sigma Chemical Co.) for 1 h at 37°C prior to toluidine blue staining.
RESULTS Parental CHO-K, cells
MATERIALS AND METHODS Cell culture CHO-K, cells were routinely grown in medium F12 supplemented with 10 % fetal-calf serum in an incubator at 37°C. 5 % COz. and 100 % humidity, as previously described [2]. Cells for the experiments presented here were removed from stocks by trypsinization and replated in medium F 12 plus 10 % extensively dialysed serum so as to have a more defined growth environment. Treated cells received 1 mM db-CAMP in the medium for a period of 16-24 h before further treatment for microscopy. Such a treatment caused the intracellular CAMP level to rise from l-2 ,L~Mto approx. 20 ,LLM[7]. As we have demonstrated previously, equal molar concentrations of the unsubstituted CAMP. sodium butvrate. adenosine, adenine, as well as various other cyclic nucleotides are inactive Il. 2. 71. Variants M-6 and M-7 are derived from &I&K,cells after treatment with ethyl methanesulfonate. Some of their properties are described in Results.
Microscopy CHO-K, and morphological variants M-6 and M-7 were grown from stock cultures at 60% confluency by trypsinization and transferred to 35-mm dishes (Falcon) containing carbon-coated coverslips and 4 ml of growth medium. When the cells had attached to the coverslips and grown to the desired density50 % for phase microscopy and 80% for electron microscopy-new medium was added, with or without 1 mM db-CAMP, and the cells were allowed to grow for an additional 16-24 h. The medium was then removed and the cells were washed twice with Puck’s Saline G [8] at 25°C. Cells were fixed for 1 h at 35°C in 3 % glutaraldehyde in 0.05 M cacodylate buffer at pH 7.2. The fixed cells were washed with Saline G and postfixed in 1 56 osmium tetroxide in 0.3 M cacodylate buffer. The cells, still attached to coverslips, were then dehydrated and infiltrated with Epon. The coverslins were quartered and placed, cell-side down, on Eponfilled Beem capsules and polymerized at 60°C. After polymerization the covershps were removed and the cells sectioned with a Porter-Blum MT-2 ultramicrotome. The sections were placed on Formvar- and carbon-coated grids, stained with uranyl acetate and
The existence of the knob-like pseudopodial structures at the periphery of the CHO cells (fig. la) has previously been demonstrated by light [3], transmission and scanning electron microscopy [5], and by time-lapse cinematography [4]. Under conditions of morphological transformation, the knobs disappear and the cells assume an elongated, fibroblast-like form with smooth membrane contours [3, 41 (fig. 2a). Toluidine blue staining demonstrates that these knobs are rich in RNA. Electron microscopic studies reveal that the knobs contain an abundant population of polyribosomes, but no other organelles. A few short, randomly oriented microtubules are present in the cytoplasm. There is a band of subplasmalemma1 microfilaments present not only in the knobs but around the entire periphery of the cell (fig. 1b). The most salient feature of morphological transformation of CHO-K, cells is their conversion to an elongated fibroblast-like form [l-3]. This conversion is prevented by agents which inhibit microtubular assembly [l, 21. In the presenceof morphological transformation agents, the microtubules elongate, increase in number, and become arrayed parallel to the long axis of the cell [6]. Microfilaments remain beneath the cell membrane (fig. 26). These observations substantiate data from previous inhibitor studies which show that one of the actions of morphological transformation agents is to promote the asExptl Cell Res 91 (1975)
424
Borman, Dumont and Hsie
Fig. 1. (a) A phase-contrast micrograph of the CHO-K, epithelial-like cells. The arrows point to phase-dense knobs at the periphery of the cells. x 1 000. (b) An electron micrograph of a knob for a CHO-K, cell. The cytoplasm of the knob consists largely of ribosomes. A band of microfilaments is present subjacent to the plasmalemma. The arrows indicate short, randomly placed microtubules. x 14 500.
sembly of microtubules and to convert the cell to the fibroblast-like form [l-3]. In the absence of morphological transformation agents, there are but a few short microtubules randomly distributed within the cytoplasm, usually in the centriolar region (see also discussions in refs [4, 51). Similar observations have been made by Porter et al. [6]. Exptl Cell Res 91 (1975)
The epithelial-like
variant, M-7
In the course of studying the induction of specific gene mutations affecting nutritional dependence and temperature sensitivity of CHO-K, cells by various physical and chemical mutagens, several morphological variant clones have been isolated. A variant, M-7, resembling the parental cell CHO-K,
CAMP, microtubules, and cell morphology
425
Fig. 2. (a) A phase-contrast micrograph of the fibroblast-like appearance of CHO-K, cells after treatment with 1 mM db-CAMP for 24 h and that the knobs have disappeared and the surface membrane is smooth. x 800. (b) An electron micrograph of the margin of a db-CAMP-treated CHO-K, cell. A band of microfilaments (MF) is present beneath the membrane. Profiles of long microtubules (MT), oriented parallel to the long axis of the cell, are also presmt. x 45 000.
cell in that it possesses a typical compact epithelial cell-like appearance with knoblike structures around the periphery has also been isolated. This variant cell line retains its epithelial-like form (fig. 3a) in the presence of morphological transformation agents for as long as 6-7 days. Despite the fact that the cells maintain their epithelial-like shape after treatment with db-CAMP, there is an apparent increase in the number and length of randomly arranged microtubules (fig. 3b). It appears as though the morphological transformation agents have promoted the assembly of microtubules in M-7 cells but not their organization into parallel arrays and the concomitant conversion of the cells to a fibroblast-like morphology.
The fibroblast-like
variant, M-6
Another variant, M-6, assumes a fibroblastlike morphology with a smooth membrane and parallel arrays of microtubules, when grown in standard basal medium, similar to the morphological transformation-agent-induced fibroblast-like form of CHO-K, cells (fig. 2a, b). Under conditions of morphological transformation, the M-6 cells become even further elongated (compare figs 2a and 4a). Exaggerated long, very thin, polar extensions which contain ribosomes, microfilaments, and parallel arrays of microtubules are observed (fig. 4b). Although an increase in the numbers of microtubules under these conditions has not been established, there is an apparent increase in the length of individual microtubules. Exptl Cell Res 91 (1975)
426 Borman, Dumont and Hsie
Fig. 3. (a) A phase contrast of a db-CAMP-treated (1 mM) M-7 epitheliallike variant cell. These cells, with or without treatment, are essentially identical with the untreated CHO-KI cells. x 800. (6) An electron micrograph of a portion of a db-CAMP-treated M-7 cell. Note the long-profile microtubules oriented randomly in the cytoplasm. x 14 500.
Exptl Cell Res 91 (1975)
CAMP, microtubules, and cell morphology
421
Fig. 4. (a) A phase-contrast micrograph cf rib-CAMP-treated (1 mM) fibroblast-like M-6 ceils. These cells become more elongated than similarly treated CHO-K, cells. x 800. (b) An electron micrograph of the elongated process of several db-CAMP-treated M-6 variant cells showing the presence of microfilaments and long microtubules in these extended processes. ’ 33 500.
DISCUSSION In mammalian cells CAMP or its derivatives appear to influence the regulation of many biochemical processes related to growth and differentiation [2, 3, 91. One of the effects of db-CAMP appears to be the promotion of microtubule assembly [I, 21. The present ultrastructural study demonstrates that under morphological transformation conditions there is an increase in the number and orientation of microtubules in CHO-K, cells, an observation also established by others ([6], see also discussions in refs [4, 51). In other cases, for example in mouse neuroblastoma cells, exposure to db-CAMP appears to stabilize the microtubules in neurites so that, during exposure to cold, the neurites do not retract into the cell body [lo]. Microtubules are ubiquitous structural
organelles of eukaryotic cells. They are regarded as playing an essential role in the maintenance of cellular morphology. The existence of random arrays of a small number of relatively short microtubules in the epithelial-like CHO-K, and M-7 cells (see figs 1b and 3b) and, on the other hand, of abundant microtubules highly organized along the long axis of either the bonafide fibroblast-like M-6 cells or the morphological transformation-agent-induced fibroblast-like CHO-K, cells (see figs 2b and 4b) provides direct evidence for the role of microtubules in conferring cell form. Although the loss of microtubule integrity has not been established, it is well known that when normal fibroblast-like, contact-inhibited cells are transformed by viruses, X-radiation, or certain chemical carcinogens they are often converted to a more epithelial-like noncontact-inhibited Exptl Cell Res 91 (1975)
428 Borman, Dumont and Hsie form [ll]. Morphologic conversion, however, which does involve the assembly and reorganization of microtubules, is associated with reversal of certain malignant characteristics of CHO cells in vitro [2-41. It thus becomes important to assess the role of microtubules, or agents which affect them, in malignant transformations. Recently, we have demonstrated that the addition of 1 mM db-CAMP to CHO-K1 cells grown under the conditions used in this study (50-80 % confluency) resulted in an increase of the endogenous level of CAMP from l-2 PM to approx. 20 ,uM, and that it remains at that level for 24 h. This increase of intracellular CAMP level appears to be due largely to the inhibitory action of intracellular N6-monobutyryl CAMP on a low Km CAMP phosphodiesterase, which results in a decreased rate of degradation of intracellular CAMP [7]. Treatment of M-6 and M-7 cells with 1 mM db-CAMP resulted in an increase of intracellular CAMP level of similar magnitude in these cells (P. O’Neill & A. Hsie. Unpublished data). The observation that, when M-7 cells are exposed to db-CAMP, the microtubules are present in increased numbers although in random orientation suggests that M-7 variant cells are capable of responding by increased microtubule assembly, but not by organizing the microtubules into ordered parallel arrays to promote fibroblastlike morphology. By the same token, the fibroblast-like M-6 cells also appear to respond by increasing the length of preexisting oriented microtubules. The experiments with M-7 cells also suggest that the assembly and the organization of microtubules are likely to be two distinct biological processes since the assembly does not necessarily lead to a change in cell form. Changes in cell form appear to be more directly dependent upon the orientation of microtubules. Studies presented in this paper suggest the Exptl Cell Res 91 (1975)
role of intracellular CAMP, through yet unknown mechanisms, in promoting microtubule assembly and organization. In view of (i) recent findings on the involvement of calcium in microtubule polymerization in vitro [12, 131 and (ii) the accumulation of evidence on the interrelationship between calcium metabolism and CAMP in many biological systems [14, 151, we have begun studies of a possible relationship between calcium and CAMP in this system. However, a more direct role of CAMP in effecting microtubule assembly in vivo cannot be excluded. The highly competent technical assistance of Patricia A. Brimerand Nina Hammer in the cell culture work, and of Carole S. King in electron microscopy, is gratefully acknowledged. We thank J. S. Cook and T. Yamada for critical review of the manuscript. L. S. B. is a predoctoral fellow of the University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, supported by grant GM 1974 from the National Institute of General Medical Sciences, NIH. Oak Ridge National Laboratory is operated by Union Carbide Corporation for the US AEC.
REFERENCES 1. Hsie. A W & Waldren. C A., J cell biol 47 (1970), 92 A: Abstr. 2. Hsie. A W & Puck. T T. Proc natl acad sci US 68 (1971) 358. ’ 3. Hsie, A W, Jones, C & Puck, T T, Proc natl acad sci US 68 (1971) 1648. 4. Puck, T T, Waldren, C A & Hsie, A W, Proc natl acad sci US 69 (1972) 1943. 5. Porter, K, Prescott, D & Frye, J, J cell biol 57 (1973) 815. 6. Porter, K R, Puck, T T, Hsie, A W & Kelley, D, Cell 2 (1974) 145. 7. Hsie, A W; Kawashima, K, O’NeilI, J P & Schriider, C H. J biol them (1974). In press. 8. Ham, R’G & Puck, T T, Methods enzymol 5 (1962) 90. 9. Kram, P, Mamont, P & Tomkins, G M, Proc natl acad sci US 70 (1973) 1432. 10. Kirkland, W L & Burton, P R, Nature new biol 240 (1972) 205. 11. Sachs, L, Curr top dev biol 2 (1967) 129. 12. Weisenberg, R C, Science 177 (1972) 1104. 13. Borisy, G G & Olmsted, J B, Science 177 (1972) 1196. 14. Rasmussen, H, Goodman, D B & Teenhouse, A, Crit rev biochem 1 (1972) 95. 15. Whitfield, J F, Rixon, R H, MacManus, J P & Balk, S D, In vitro 8 (1973) 257. Received July 4, 1974
