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Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer.

Havaki S, Kouloukoussa M, Amawi K, Drosos Y, Arvanitis LD, Goutas N, Vlachodimitropoulos D, Vassilaros SD, Katsantoni EZ, Voloudakis-Baltatzis I, Aleporou-Marinou V, Kittas C, Marinos E - Cancer Cell Int. (2007)

Bottom Line: In the present study, short-term primary breast cancer cell cultures were developed.In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells.Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Histology and Embryology, Medical School, University of Athens, 75 Mikras Asias Str, Goudi, Greece. shavaki@med.uoa.gr

ABSTRACT

Background: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting.

Results: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells.

Conclusion: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

No MeSH data available.


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Representative Western immunoblots for integrin beta3 subunit and actin on whole cell extracts of MDA-MB-435 cell line (lane 1), normal breast tissue primary culture cells (lane 2) and primary culture cells of infiltrating breast carcinomas (grade II) (lanes 3, 4, 5). Equal amounts of protein (40 μg) were loaded from each sample and subjected to Western blot analysis. Integrin beta3 subunit was not detected in normal breast tissue cells, while differential expression of beta3 was observed in breast cancer cell samples. In the latter, a higher expression of actin was also observed in comparison with that of normal breast tissue cells.
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Figure 7: Representative Western immunoblots for integrin beta3 subunit and actin on whole cell extracts of MDA-MB-435 cell line (lane 1), normal breast tissue primary culture cells (lane 2) and primary culture cells of infiltrating breast carcinomas (grade II) (lanes 3, 4, 5). Equal amounts of protein (40 μg) were loaded from each sample and subjected to Western blot analysis. Integrin beta3 subunit was not detected in normal breast tissue cells, while differential expression of beta3 was observed in breast cancer cell samples. In the latter, a higher expression of actin was also observed in comparison with that of normal breast tissue cells.

Mentions: Western immunoblotting of integrin beta3 assessed quantitatively the expression of integrin alphavbeta3 in breast cancer cells vs. normal breast cells. In parallel, the level of actin expression was examined in the same samples. Human breast carcinoma cell line MDA-MB-435 was used as positive control, since it highly expresses this integrin alphavbeta3 [24]. Equal amounts of protein (40 μg) were loaded from each sample and the expression levels of integrin beta3 subunit and of actin were studied. No detectable amounts of integrin beta3 subunit were observed in normal breast tissue primary culture cells (Fig. 7, lane 2). However, integrin beta3 subunit was expressed at various levels in breast cancer cells derived from different biopsies of infiltrating breast carcinomas with the same histological grade (grade II) (Fig. 7, lanes 3, 4, 5), confirming the heterogeneity characterizing breast cancer. In fact, one of the breast cancer samples (Fig. 7, lane 5) expressed the integrin beta3 at approximately the same high level of that of MDA-MB-435 cell line (Fig. 7, lane 1). On the other hand, actin was expressed at lower levels in normal breast cells (Fig. 7, lane 2) compared to the higher expression levels that were observed in breast cancer cells from primary cultures (Fig. 7, lanes 3, 4, 5). The latter were almost the same with that expressed in the MDA-MB-435 cell line (Fig. 7, lane 1).


Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer.

Havaki S, Kouloukoussa M, Amawi K, Drosos Y, Arvanitis LD, Goutas N, Vlachodimitropoulos D, Vassilaros SD, Katsantoni EZ, Voloudakis-Baltatzis I, Aleporou-Marinou V, Kittas C, Marinos E - Cancer Cell Int. (2007)

Representative Western immunoblots for integrin beta3 subunit and actin on whole cell extracts of MDA-MB-435 cell line (lane 1), normal breast tissue primary culture cells (lane 2) and primary culture cells of infiltrating breast carcinomas (grade II) (lanes 3, 4, 5). Equal amounts of protein (40 μg) were loaded from each sample and subjected to Western blot analysis. Integrin beta3 subunit was not detected in normal breast tissue cells, while differential expression of beta3 was observed in breast cancer cell samples. In the latter, a higher expression of actin was also observed in comparison with that of normal breast tissue cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2116995&req=5

Figure 7: Representative Western immunoblots for integrin beta3 subunit and actin on whole cell extracts of MDA-MB-435 cell line (lane 1), normal breast tissue primary culture cells (lane 2) and primary culture cells of infiltrating breast carcinomas (grade II) (lanes 3, 4, 5). Equal amounts of protein (40 μg) were loaded from each sample and subjected to Western blot analysis. Integrin beta3 subunit was not detected in normal breast tissue cells, while differential expression of beta3 was observed in breast cancer cell samples. In the latter, a higher expression of actin was also observed in comparison with that of normal breast tissue cells.
Mentions: Western immunoblotting of integrin beta3 assessed quantitatively the expression of integrin alphavbeta3 in breast cancer cells vs. normal breast cells. In parallel, the level of actin expression was examined in the same samples. Human breast carcinoma cell line MDA-MB-435 was used as positive control, since it highly expresses this integrin alphavbeta3 [24]. Equal amounts of protein (40 μg) were loaded from each sample and the expression levels of integrin beta3 subunit and of actin were studied. No detectable amounts of integrin beta3 subunit were observed in normal breast tissue primary culture cells (Fig. 7, lane 2). However, integrin beta3 subunit was expressed at various levels in breast cancer cells derived from different biopsies of infiltrating breast carcinomas with the same histological grade (grade II) (Fig. 7, lanes 3, 4, 5), confirming the heterogeneity characterizing breast cancer. In fact, one of the breast cancer samples (Fig. 7, lane 5) expressed the integrin beta3 at approximately the same high level of that of MDA-MB-435 cell line (Fig. 7, lane 1). On the other hand, actin was expressed at lower levels in normal breast cells (Fig. 7, lane 2) compared to the higher expression levels that were observed in breast cancer cells from primary cultures (Fig. 7, lanes 3, 4, 5). The latter were almost the same with that expressed in the MDA-MB-435 cell line (Fig. 7, lane 1).

Bottom Line: In the present study, short-term primary breast cancer cell cultures were developed.In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells.Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Histology and Embryology, Medical School, University of Athens, 75 Mikras Asias Str, Goudi, Greece. shavaki@med.uoa.gr

ABSTRACT

Background: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting.

Results: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells.

Conclusion: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

No MeSH data available.


Related in: MedlinePlus