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Distribution of actin filaments in human malignant keratinocytes
Cell Biology
International
Reports,
Vol. 72, No. 3, March
DISTRIBUTION OF ACTIN FILAMENTS HUMAN MALIGNANT KERATINOCYTES Y. Department of Medicine,
of
Dermatology, Osaka 553,
189
1988
IN
Kitano Osaka JAPAN
University
School
ABSTRACT Distribution of actin filaments in human malignant keratinocytes was examined by immunofluorescence staining. The primary cultures were obtained from a squamous a basal cell carcinoma, and Bowen's discell carcinoma, ease. Rhodamine-phalloid'in staining revealed that actin filaments were occasionally organized to form stress fibers, many short bundles with a ripple appearance, Some of these and regular arrays of actin patches. structures appeared in untransformed keratinocytes as a result of a brief exposure to a tumor promotor, These findings suggest that regulation of actin TPA. functions is involved in neoplastic processes from the very early stages and that alteration is persistent in neoplastic cells. INTRODUCTION It is widely believed that many changes in the cytoskeletal system must accompany transformation of normal The clarification of these cells to neoplastic cells. events would be an important step toward understanding the mechanisms of neoplastic transformation leading to malignancy. prominent changes in the In fibroblastic cells, organization of actin filaments were observed in their neoplastic counterparts (Pollack et al., 1975, Wang and Goldberg, 1976). A potent tumor promotor, 12-0tetradecanoylphorbol-13-acetate (TPA), induced a rapid redistribution of actin filaments, and a loss of the ordered actin-containing structures in normal fibroblasts (Rifkin et al., 1979). Recently, we reported our observation of changes of actin distribution in one of the most differentiated human keratinocytes, epithelial cells, after treatment with TPA (Kitano et al., 1986). In normal human keratinocytes, distribution of actin filaments was diffuse and TPA induced many short bundles of actin, and large actin-containing 0309-1651188/030189-61$03.00/0
Q1988AcademicPressLtd
Cell Biology
ribbons. To extend actin distribution MATERIALS
International
in
Reports,
these observations, some skin tumor
Vol. 72, No. 3, March
we examined cells.
1988
the
AND METHODS
Cell culture. Tumor cells were obtained from a squamous cell carcinoma of a 55-year old male, from Bowen's disease in a 77-year old female and from a basal cell carcinoma of a 67-year old female. The tumors were excised surgically and were soaked in Eagles MEM containing 500 U/ml dispase (Godo Shusei) (Kitano and Okada, 1983). Following this treatment for about were separated from the underly24 h at 4OC, the tumors ing dermis without fragmentation and a suspension of the tumor cells was obtained by treatment with 0.25% trypsin for 5 min at 37OC. The cells were suspended in Eagle's MEM supplemented with 20% fetal bovine serum and were grown on glass coverslips. Immunofluorescence microscopy. The cells grown on glass coverslips were washed with phosphate buffered saline (PBS). For actin staining, the cells were fixed with 3.7% formaldehyde in PBS for 20 min at room temperature, treated with 0.2% Triton X-100 in PBS for 5 min and incubated with rhodamine-phalloidin (Molecular Probes Inc.) for 20 min. For keratin staining, the cells were fixed with absolute methanol for 10 min at 4'C, incubated with antikeratin antibody (DAKO) and stained with fluorescein-conjugated goat antirabbit IgG (Medical and Biological Inc.). Between treatments, the samples were Microscopic examination washed with PBS three times. was carried out with an Olympus fluorescence microscope BH-2 after mounting in Perma Fluor (Lipshaw/Immunon). RESULTS Cells of squamous cell carcinoma grew mostly connected with each other to form a small sheet (Fig. 1). Singly isolated cells, however, were frequently The cells were polymorphic and varied in observed. size with a nucleus having a few prominent nucleoli. Sometimes large multipolar or ameboid cells with one or several nuclei were observed at the margin of the These were identified as stromal cells because sheet. of negative staining to antikeratin antibody. Rhodamine-phalloidin staining revealed that actin filaments were diffusely distributed or formed fine, complex meshes in most of the cells. Those containing a fine, branching network occasionally had several focal The actin filaments were frequently centers (Fig. 2). organized into structures which were not observed in untransformed keratinocytes. Figure 3 shows many short bundles of actin filaments with a ripple appearance
Cell Biology
Fig.
1.
fnternational
Reports,
Cells of squamous cell carcinoma in culture. 7-day-culture. Phase contrast. Bar 10 pm.
Vol. 72, No. 3, March
Fig.
2.
1988
191
Rhodamine-phalloidin staining of cells of squamous cell carcinoma. Fine network of actin filaments converges at several points. 7-dayculture. Bar 1 Urn.
which was a prominent feature seen shortly after the addition of TPA (Kitano et al., 1986). Some cells had short but distinct arrays of stress fibers (Fig. 4). Regular arrays of actin patches, found by Carley et al, were also observed (Fig. 5) (Carley et al., 1981), while large and irregular actin patches were sometimes seen. These actin patches were mostly observed in isolated cells. The cells of the basal cell carcinoma had essentially the same organization of actin filaments as those of the squamous cell carcinoma. A somewhat different assemblage of actin filaments was observed in the cells of Bowen's disease. Figure 6 shows actin bundles along the rim of the cells and short stress fibers of random direction. In other parts of these cells, hardly any actin fibers appeared. Regular arrays of actin patches, however, were frequently observed in the cells of Bowen's disease (Fig. 5). DISCUSSION Actin structures in cells of malignant skin tumors of epidermal origin were stained. Although actin filaments rarely assemble to form stress fibers in untransformed human keratinocytes, stress fibers were seen frequently in these malignant keratinocytes. Since stress fibers are closely related to attachment to the substrate and cellular movement (Wehland et al., 1979, Wang, 1984, Meigs and Wang, 1986), there should exist a difference
Cell Biology
192
Fig.
3.
Fig.
4.
Fig.
5.
Fig.
6.
International
Reports,
Vol. 12, No. 3, March
Rhodamine-phalloidin staining squamous cell carcinoma. Fine, assemblage of actin filaments. Bar 1 urn. Rhodamine-phalloidin staining squamous cell carcinoma. Array stress fibers. 8-day-culture. Rhodamine-phalloidin staining Bowen's disease. Regular array patches, and large, irregular 7-day-culture. Bar 1 urn. Rhodamine-phalloidin staining Bowen's disease. 5-day-culture.
of
1988
cells of ripple-like 8-day-culture.
of
cells of of short Bar 2 urn. of cells of of actin actin patches. of
cells Bar
of 2 vrn.
in attachment and movement between untransformed and malignant keratinocytes. But the difference is not the same as that observed in fibroblastic cells in which actin-containing stress fibers disappear after treatment (Rifkin et with a tumor promotor or an oncogenic virus al., 1979, Edelman and Yahara, 1976, Wang and Goldberg, 1976) - In keratinocytes, TPA induced assemblage of
Cell Biology
International
Reports,
Vol. 12, No. 3, March
193
1988
actin filaments in the form of ripples or thick ribbon-like structures (Kitano, et al., 1986). It is interesting that these structures were also observed in malignant keratinocytes. Regular arrays of actin patches found in transformed cells were observed, but only rarely (Carley et al., 1981, Stickel and Wang, 1987) . Cutaneous squamous cell carcinoma invades adjacent tissue destructively and metastasizes. Basal cell carcinoma shows destructive invasion but rarely Bowen's disease is an intraepidermal metastasizes. squamous cell carcinoma and represents biologically a precancerous dermatosis. As for the distribution and organization of actin filaments, there was no essential difference among these three carcinomas. These findings, together with those induced by TPA, suggest that regulation of actin functions is involved in neoplastic process from the very early stages and that alteration is persistent in neoplastic cells. REFERENCES Carley, W.W., Barak, L.S. and Webb, W.W. (1981) F-actin aggregates in transformed cells. The Journal of Cell Biology, 90, 797-802. Edelman, G.M. and Yahara, I. (1976) Temperature-sensitive changes in surface modulating assemblies of fibroblasts transformed by mutants of Rous sarcoma virus. Proceedings of the National Academy of Sciences of the USA, 73, 2047-2051. Kitano, Y. and Okada, N. (1983) Separation of epidermal sheet by dispase. British Journal of Dermatology, 108,
555-560.
KitanO, Y., Okada, N. and Adachi, J. (1986) TPA-induced alteration of actin organization in cultured human keratinocytes. Experimental Cell Research, 167, 369-375.
Meigs, J.B. alpha-actinin in living
and
Wang, Y.-L. (1986) Reorganization and vinculin induced by a phorbol cells. The Journal of Cell Biology,
of ester 102,
1430-1438.
Pollack, R., Osborn, M. and Weber, K. (1975) Patterns of organization of actin and myosin in normal and transformed cultured cells. Proceedings of the National Academy of Sciences of the USA, 72, 994-998. Rifkin, D.B., Crowe, R.M. and Pollack, R. (1979) Tumor promotors induce changes in the chick embryo fibroblasts cytoskeleton. Cell, 18, 361-368. Stickel, S.K. and Wang, Y.-L. (1987) Alpha-actinincontaining aggregates in transformed cells are highly dynamic structures. The Journal of Cell Biology, 104, 1521-1526.
194
Cell Biology
International
Reports,
Vol. 12, No. 3, March
1988
Wang, Y.-L. (1984) Reorganization of actin filament bundles in living fibroblasts. The Journal of Cell Biology, 99, 1478-1485. Wang, Y.-L. and Goldberg, A.R. (1976) Changes in microfilament organization and surface topography upon transformation of chick embryo fibroblasts with Rous sarcoma virus. Proceedings of the National Academy of Sciences of the USA, 73, 4065-4069. Wehland, J., Osborn, M. and Weber, K. (1979) Cellto-substratum contacts in living cells: A direct correlation between interference-reflexion and indirect-immunofluorescence microscopy using antibodies against actin and a-actinin. The Journal of Cell Science, 37, 257-273.
Received:
2.11.87
Accepted:
12.1.88
International
Reports,
Vol. 72, No. 3, March
DISTRIBUTION OF ACTIN FILAMENTS HUMAN MALIGNANT KERATINOCYTES Y. Department of Medicine,
of
Dermatology, Osaka 553,
189
1988
IN
Kitano Osaka JAPAN
University
School
ABSTRACT Distribution of actin filaments in human malignant keratinocytes was examined by immunofluorescence staining. The primary cultures were obtained from a squamous a basal cell carcinoma, and Bowen's discell carcinoma, ease. Rhodamine-phalloid'in staining revealed that actin filaments were occasionally organized to form stress fibers, many short bundles with a ripple appearance, Some of these and regular arrays of actin patches. structures appeared in untransformed keratinocytes as a result of a brief exposure to a tumor promotor, These findings suggest that regulation of actin TPA. functions is involved in neoplastic processes from the very early stages and that alteration is persistent in neoplastic cells. INTRODUCTION It is widely believed that many changes in the cytoskeletal system must accompany transformation of normal The clarification of these cells to neoplastic cells. events would be an important step toward understanding the mechanisms of neoplastic transformation leading to malignancy. prominent changes in the In fibroblastic cells, organization of actin filaments were observed in their neoplastic counterparts (Pollack et al., 1975, Wang and Goldberg, 1976). A potent tumor promotor, 12-0tetradecanoylphorbol-13-acetate (TPA), induced a rapid redistribution of actin filaments, and a loss of the ordered actin-containing structures in normal fibroblasts (Rifkin et al., 1979). Recently, we reported our observation of changes of actin distribution in one of the most differentiated human keratinocytes, epithelial cells, after treatment with TPA (Kitano et al., 1986). In normal human keratinocytes, distribution of actin filaments was diffuse and TPA induced many short bundles of actin, and large actin-containing 0309-1651188/030189-61$03.00/0
Q1988AcademicPressLtd
Cell Biology
ribbons. To extend actin distribution MATERIALS
International
in
Reports,
these observations, some skin tumor
Vol. 72, No. 3, March
we examined cells.
1988
the
AND METHODS
Cell culture. Tumor cells were obtained from a squamous cell carcinoma of a 55-year old male, from Bowen's disease in a 77-year old female and from a basal cell carcinoma of a 67-year old female. The tumors were excised surgically and were soaked in Eagles MEM containing 500 U/ml dispase (Godo Shusei) (Kitano and Okada, 1983). Following this treatment for about were separated from the underly24 h at 4OC, the tumors ing dermis without fragmentation and a suspension of the tumor cells was obtained by treatment with 0.25% trypsin for 5 min at 37OC. The cells were suspended in Eagle's MEM supplemented with 20% fetal bovine serum and were grown on glass coverslips. Immunofluorescence microscopy. The cells grown on glass coverslips were washed with phosphate buffered saline (PBS). For actin staining, the cells were fixed with 3.7% formaldehyde in PBS for 20 min at room temperature, treated with 0.2% Triton X-100 in PBS for 5 min and incubated with rhodamine-phalloidin (Molecular Probes Inc.) for 20 min. For keratin staining, the cells were fixed with absolute methanol for 10 min at 4'C, incubated with antikeratin antibody (DAKO) and stained with fluorescein-conjugated goat antirabbit IgG (Medical and Biological Inc.). Between treatments, the samples were Microscopic examination washed with PBS three times. was carried out with an Olympus fluorescence microscope BH-2 after mounting in Perma Fluor (Lipshaw/Immunon). RESULTS Cells of squamous cell carcinoma grew mostly connected with each other to form a small sheet (Fig. 1). Singly isolated cells, however, were frequently The cells were polymorphic and varied in observed. size with a nucleus having a few prominent nucleoli. Sometimes large multipolar or ameboid cells with one or several nuclei were observed at the margin of the These were identified as stromal cells because sheet. of negative staining to antikeratin antibody. Rhodamine-phalloidin staining revealed that actin filaments were diffusely distributed or formed fine, complex meshes in most of the cells. Those containing a fine, branching network occasionally had several focal The actin filaments were frequently centers (Fig. 2). organized into structures which were not observed in untransformed keratinocytes. Figure 3 shows many short bundles of actin filaments with a ripple appearance
Cell Biology
Fig.
1.
fnternational
Reports,
Cells of squamous cell carcinoma in culture. 7-day-culture. Phase contrast. Bar 10 pm.
Vol. 72, No. 3, March
Fig.
2.
1988
191
Rhodamine-phalloidin staining of cells of squamous cell carcinoma. Fine network of actin filaments converges at several points. 7-dayculture. Bar 1 Urn.
which was a prominent feature seen shortly after the addition of TPA (Kitano et al., 1986). Some cells had short but distinct arrays of stress fibers (Fig. 4). Regular arrays of actin patches, found by Carley et al, were also observed (Fig. 5) (Carley et al., 1981), while large and irregular actin patches were sometimes seen. These actin patches were mostly observed in isolated cells. The cells of the basal cell carcinoma had essentially the same organization of actin filaments as those of the squamous cell carcinoma. A somewhat different assemblage of actin filaments was observed in the cells of Bowen's disease. Figure 6 shows actin bundles along the rim of the cells and short stress fibers of random direction. In other parts of these cells, hardly any actin fibers appeared. Regular arrays of actin patches, however, were frequently observed in the cells of Bowen's disease (Fig. 5). DISCUSSION Actin structures in cells of malignant skin tumors of epidermal origin were stained. Although actin filaments rarely assemble to form stress fibers in untransformed human keratinocytes, stress fibers were seen frequently in these malignant keratinocytes. Since stress fibers are closely related to attachment to the substrate and cellular movement (Wehland et al., 1979, Wang, 1984, Meigs and Wang, 1986), there should exist a difference
Cell Biology
192
Fig.
3.
Fig.
4.
Fig.
5.
Fig.
6.
International
Reports,
Vol. 12, No. 3, March
Rhodamine-phalloidin staining squamous cell carcinoma. Fine, assemblage of actin filaments. Bar 1 urn. Rhodamine-phalloidin staining squamous cell carcinoma. Array stress fibers. 8-day-culture. Rhodamine-phalloidin staining Bowen's disease. Regular array patches, and large, irregular 7-day-culture. Bar 1 urn. Rhodamine-phalloidin staining Bowen's disease. 5-day-culture.
of
1988
cells of ripple-like 8-day-culture.
of
cells of of short Bar 2 urn. of cells of of actin actin patches. of
cells Bar
of 2 vrn.
in attachment and movement between untransformed and malignant keratinocytes. But the difference is not the same as that observed in fibroblastic cells in which actin-containing stress fibers disappear after treatment (Rifkin et with a tumor promotor or an oncogenic virus al., 1979, Edelman and Yahara, 1976, Wang and Goldberg, 1976) - In keratinocytes, TPA induced assemblage of
Cell Biology
International
Reports,
Vol. 12, No. 3, March
193
1988
actin filaments in the form of ripples or thick ribbon-like structures (Kitano, et al., 1986). It is interesting that these structures were also observed in malignant keratinocytes. Regular arrays of actin patches found in transformed cells were observed, but only rarely (Carley et al., 1981, Stickel and Wang, 1987) . Cutaneous squamous cell carcinoma invades adjacent tissue destructively and metastasizes. Basal cell carcinoma shows destructive invasion but rarely Bowen's disease is an intraepidermal metastasizes. squamous cell carcinoma and represents biologically a precancerous dermatosis. As for the distribution and organization of actin filaments, there was no essential difference among these three carcinomas. These findings, together with those induced by TPA, suggest that regulation of actin functions is involved in neoplastic process from the very early stages and that alteration is persistent in neoplastic cells. REFERENCES Carley, W.W., Barak, L.S. and Webb, W.W. (1981) F-actin aggregates in transformed cells. The Journal of Cell Biology, 90, 797-802. Edelman, G.M. and Yahara, I. (1976) Temperature-sensitive changes in surface modulating assemblies of fibroblasts transformed by mutants of Rous sarcoma virus. Proceedings of the National Academy of Sciences of the USA, 73, 2047-2051. Kitano, Y. and Okada, N. (1983) Separation of epidermal sheet by dispase. British Journal of Dermatology, 108,
555-560.
KitanO, Y., Okada, N. and Adachi, J. (1986) TPA-induced alteration of actin organization in cultured human keratinocytes. Experimental Cell Research, 167, 369-375.
Meigs, J.B. alpha-actinin in living
and
Wang, Y.-L. (1986) Reorganization and vinculin induced by a phorbol cells. The Journal of Cell Biology,
of ester 102,
1430-1438.
Pollack, R., Osborn, M. and Weber, K. (1975) Patterns of organization of actin and myosin in normal and transformed cultured cells. Proceedings of the National Academy of Sciences of the USA, 72, 994-998. Rifkin, D.B., Crowe, R.M. and Pollack, R. (1979) Tumor promotors induce changes in the chick embryo fibroblasts cytoskeleton. Cell, 18, 361-368. Stickel, S.K. and Wang, Y.-L. (1987) Alpha-actinincontaining aggregates in transformed cells are highly dynamic structures. The Journal of Cell Biology, 104, 1521-1526.
194
Cell Biology
International
Reports,
Vol. 12, No. 3, March
1988
Wang, Y.-L. (1984) Reorganization of actin filament bundles in living fibroblasts. The Journal of Cell Biology, 99, 1478-1485. Wang, Y.-L. and Goldberg, A.R. (1976) Changes in microfilament organization and surface topography upon transformation of chick embryo fibroblasts with Rous sarcoma virus. Proceedings of the National Academy of Sciences of the USA, 73, 4065-4069. Wehland, J., Osborn, M. and Weber, K. (1979) Cellto-substratum contacts in living cells: A direct correlation between interference-reflexion and indirect-immunofluorescence microscopy using antibodies against actin and a-actinin. The Journal of Cell Science, 37, 257-273.
Received:
2.11.87
Accepted:
12.1.88