Skip to main content

Advertisement

SpringerLink
Log in

Nuclear patterns of human breast cancer cells during apoptosis: characterisation by fractal dimension and co-occurrence matrix statistics

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

An analytical strategy combining fractal geometry and grey-level co-occurrence matrix (GLCM) statistics was devised to investigate ultrastructural changes in oestrogen-insensitive SK-BR3 human breast cancer cells undergoing apoptosis in vitro. Apoptosis was induced by 1 μM calcimycin (A23187 Ca2+ ionophore) and assessed by measuring conventional cellular parameters during the culture period. SK-BR3 cells entered the early stage of apoptosis within 24 h of treatment with calcimycin, which induced detectable changes in nuclear components, as documented by increased values of most GLCM parameters and by the general reduction of the fractal dimensions. In these affected cells, morphonuclear traits were accompanied by the reduction of distinct gangliosides and loss of unidentifiable glycolipid molecules at the cell surface. All these changes were shown to be involved in apoptosis before the detection of conventional markers, which were only measurable during the active phases of apoptotic cell death. In overtly apoptotic cells treated with 1 μM calcimycin for 72 h, most nuclear components underwent dramatic ultrastructural changes, including marginalisation and condensation of chromatin, as reflected in a significant reduction of their fractal dimensions. Hence, both fractal and GLCM analyses confirm that the morphological reorganisation of nuclei, attributable to a loss of structural complexity, occurs early in apoptosis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

FD:

fractal dimension

GLCM:

grey-level co-occurrence matrix

FITC:

fluorescein isothiocyanate

PI:

propidium iodide

TLE:

total lipid extract

NM:

perinuclear membrane

NMBHC:

nuclear membrane-bound heterochromatin space and outline

SHC:

scattered heterochromatin space and outline

HC:

total heterochromatin space and outline

IC:

inter(eu)chromatin space and outline

TdT:

Terminal deoxynucleotidyl Transferase

References

  • Baish JW, Jain RK (2000) Fractals and cancer. Cancer Res 60:3683–3688

    PubMed  CAS  Google Scholar 

  • Baumann G, Barth A, Nonnenmacher TF (1994) Measuring fractal dimension of cell contours: practical approaches and their limitations. In: Nonnenmacher TF, Losa GA, Merlini D, Weibel ER (eds) Fractals in biology and medicine, vol 1. Birkhäuser, Basel Boston Berlin, pp 182–189

    Google Scholar 

  • Bernard F, Bossu JL, Gaillard S (2001) Identification of living oligodendrocyte developmental stages by fractal analysis of cell morphology. J Neurosci Res 65:439–445

    Article  PubMed  CAS  Google Scholar 

  • Bianciardi G, Miracco C, Santi MD, Perrone A, Bruni A, Luzi P (2002) Fractal dimension of lymphocytic nuclear membrane in Mycosis fungoides and chronic dermatitis. In: Losa GA, Merlini D, Nonnenmacher TF, Weibel ER (eds) Fractals in biology and medicine, vol 3. Birkhäuser, Basel Boston Berlin pp 150–155

    Google Scholar 

  • Biggiogera M, Bottone MG, Pellicciari C (1997) Nuclear ribonucleoprotein-containing structures undergo severe rearrangement during spontaneous thymocyte apoptosis. A morphological study by electron microscopy. Histochem Cell Biol 107:331–336

    Article  PubMed  CAS  Google Scholar 

  • Carstensen JM (1992) Description and simulation of visual texture. Institute of Mathematical Statistics and Operations Research, Technical University of Denmark, Lyngby, Denmark

    Google Scholar 

  • Castelli C (2001) Apoptotic cell death in human breast cancer cells: characterisation of functional pathways and fractal-statistical image analysis of cellular ultrastructures. PhD Thesis. University of Lausanne, Lausanne, Switzerland

  • Castelli C, Losa GA (2001) Ultrastructural complexity of nuclear components during early apoptotic phases in breast cancer cells. Anal Cell Pathol 23:1–9

    PubMed  CAS  Google Scholar 

  • Chan KL (1995) Quantitative characterization of electron micrograph image using fractal feature. IEEE Trans Biomed Eng 42:1033–1037

    Article  PubMed  CAS  Google Scholar 

  • Conners RW, Trivedi MM, Harlow CA (1984) Segmentation of a high-resolution urban scene. Comput Vis Graph Image Process 25:273–310

    Article  Google Scholar 

  • Cornelissen M, Philippé J, De Sitter S, De Ridder L (2002) Annexin V expression in apoptotic peripheral blood lymphocytes: an electron microscopic evaluation. Apoptosis 7:41–47

    Article  PubMed  CAS  Google Scholar 

  • Cross SS (1994) The application of fractal geometric analysis to microscopic images. Micron 5:101–113

    Article  Google Scholar 

  • De Maria R, Lenti L, Malisan F, d'Agostino F, Tomassini B, Zeuner A, Rippo MR, Testi R (1997) Requirement for GD3 ganglioside in CD-95 and ceramide-induced apoptosis. Science 277:1652–1655

    Article  PubMed  Google Scholar 

  • De Vico G, Peretti V, Losa GA (2005) Fractal organization of feline oocyte cytoplasm. Eur J Histochem 49(2):151–156

    PubMed  Google Scholar 

  • Del Bino G, Darzynkiewicz Z, Degraef C, Mosselmans R, Fokan D, Galand P (1999) Comparison of methods based on annexin-V binding, DNA content or TUNEL for evaluating cell death in HL-60 and adherent MCF-7 cells. Cell Prolif 32:25–37

    Article  PubMed  CAS  Google Scholar 

  • Dioguardi N, Grizzi F (2002) Fractal dimension exponent for quantitative evaluation of liver extracellular matrix in biopsy specimens. In: Losa GA, Merlini D, Nonnenmacher TF, Weibel ER (eds) Fractals in biology and medicine, vol 3. Birkhäuser, Basel Boston Berlin, pp 113–120

    Google Scholar 

  • Dollinger JW, Metzler R, Nonnenmacher TF (1998) Bi-asymptotic fractals: fractals between lower and upper bounds. J Phys Math Gen 31:3839–3847

    Article  Google Scholar 

  • Doudkine A, MacAulay C, Poulin N, Palcic B (1995) Nuclear texture measurements in image cytometry. Pathologica 87:286–299

    PubMed  CAS  Google Scholar 

  • Einstein AJ, Wu HS, Sanchez M, Gil J (1998) Fractal characterization of chromatin appearance for diagnosis in breast cytology. J Pathol 185:366–381

    Article  PubMed  CAS  Google Scholar 

  • Falcieri E, Gobbi P, Zamai L, Vitale M (1994a) Ultrastructural features of apoptosis. Scanning Microsc 8:653–666

    PubMed  CAS  Google Scholar 

  • Falcieri E, Zamai L, Santi S, Cinti C, Gobbi P, Bosco D, Cataldi A, Betts C, Vitale M (1994b) The behaviour of nuclear domains in the course of apoptosis. Histochemistry 102:221–231

    Article  PubMed  CAS  Google Scholar 

  • Ferguson DJP, Anderson TJ (1981) Ultrastructural observation on cell death by apoptosis in the resting human breast. Virchows Arch 393:193–203

    Article  CAS  Google Scholar 

  • Glasbey CA, Horgan GW (1995) Image analysis for the biological Sciences. In: Barnett V (ed) Statistics in practice. Wiley, Chichester

    Google Scholar 

  • Gorczyca W, Gong J, Darzynkiewicz Z (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assay. Cancer Res 53:1945–1951

    PubMed  CAS  Google Scholar 

  • Graber R, Losa GA (1995) Changes in the activities of signal transduction and transport membrane enzymes in CEM lymphoblastoid cells by glucocorticoid-induced apoptosis. Anal Cell Pathol 8:159–176

    PubMed  CAS  Google Scholar 

  • Gwag BJ, Canzoniero LM, Sensi SL, Demaro JA, Koh JY, Goldberg MP, Jacquin M, Choi DW (1999) Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons. Neuroscience 90:1339–1348

    Article  PubMed  CAS  Google Scholar 

  • Haralick RM, Shanmugam K, Dinstein IH (1973) Textural features for image classification. IEEE Trans Syst Man Cybern 69:610–621

    Article  Google Scholar 

  • Irinopoulou T, Rigaut JP, Benson MC (1993) Toward objective prognostic grading of prostatic carcinoma using image analysis. Anal Quant Cytol Histol 15:341–414

    PubMed  CAS  Google Scholar 

  • Kerr JFR, Wyllie AH, Currie R (1972) Apoptosis: a basic biological phenomenon with wide-ranging implication in tissue kinetics. Br J Cancer 26:239–257

    PubMed  CAS  Google Scholar 

  • Kerr JFR, Winterforth CM, Harmon BV (1994) Apoptosis. It's significant in cancer and cancer therapy. Cancer 73:2013–2026

    Article  PubMed  CAS  Google Scholar 

  • Kittler J, Illingworth J (1986) Minimum error thresholding. Pattern Recogn 19:41–47

    Article  Google Scholar 

  • Ladisch S, Gillard B (1985) A solvent partition method for microscale ganglioside purification. Anal Biochem 146:220–231

    Article  PubMed  CAS  Google Scholar 

  • Landini G, Rigaut JP (1997) A method for estimating the dimension of asymptotic fractal sets. Bioimaging 5:50–70

    Article  Google Scholar 

  • Landini G, Rippin JW (1993) Fractal dimension of epithelial-connective tissue interfaces in premalignant and malignant epithelial lesions of the floor of the mouth. Anal Quant Cytol Histol 15:144–151

    PubMed  CAS  Google Scholar 

  • Landini G, Rippin JW (1996) Quantification of nuclear pleomorphism using asymptotic fractal model. Anal Quant Cytol Histol 18:167–176

    PubMed  CAS  Google Scholar 

  • Losa GA (1986) Plasma membrane properties in human breast tissues and cell lines. Ann N Y Acad Sci 464:431–435

    Article  Google Scholar 

  • Losa GA, Graber R (1998) Apoptotic cell death and the proliferative capacity of human breast cancer. Anal Cell Pathol 16:1–10

    PubMed  CAS  Google Scholar 

  • Losa GA, Nonnenmacher TF (1996) Self-similarity and fractal irregularity in pathologic tissues. Modern Pathol 9:174–182

    CAS  Google Scholar 

  • Losa GA, Baumann G, Nonnenmacher TF (1992) Fractal dimension of pericellular membranes in human lymphocytes and lymphoblastic leukemic cells. Pathol Res Pract 188:680–686

    PubMed  CAS  Google Scholar 

  • Losa GA, Graber R, Baumann G, Nonnenmacher TF (1999) Effects of steroid hormones on nuclear membrane and membrane-bound heterochromatin from breast cancer cells evaluated by fractal morphometry. Anal Quant Cytol Histol 21:430–436

    PubMed  CAS  Google Scholar 

  • MacAulay C, Palcic B (1990) Fractal texture features based on optical density surface area. Use in image analysis of cervical cells. Anal Quant Cytol Histol 12:394–398

    PubMed  CAS  Google Scholar 

  • Mandelbrot BB (1983) The fractal geometry of the nature. Freeman, San Francisco

    Google Scholar 

  • Maraldi NM, Zini N, Santi S, Ognibene A, Rizzoli R, Mazzotti G, Manzoli FA (1998) Cytochemistry of the functional domains of the nucleus in normal and in pathologic conditions. Eur J Histochem 42:41–53

    PubMed  Google Scholar 

  • Marinelli F, Santoro R, Maraldi NM (1998) Fractal analysis of heterochromatin in nuclear domains in lymphocytes. In: Losa GA, Merlini D, Nonnenmacher TF, Weibel ER (eds) Fractals in biology and medicine, vol 2. Birkhäuser, Basel Boston Berlin, pp 220–225

    Google Scholar 

  • Marquina G, Waki H, Fernandez LE, Kon K, Carr A, Valiente O, Perez R, Ando S (1996) Gangliosides expressed in human breast cancer. Cancer Res 56:5165–5171

    PubMed  CAS  Google Scholar 

  • Müthing J (1996) High resolution thin-layer chromatography of gangliosides. J Chromatogr A 720:3–25

    Article  PubMed  Google Scholar 

  • Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279

    Article  PubMed  CAS  Google Scholar 

  • Nielsen B, Albregtsen F, Danielsen HE (1999) The use of fractal features from the periphery of cell nuclei as a classification tool. Anal Cell Pathol 19:21–37

    PubMed  CAS  Google Scholar 

  • Nohara K, Wang F, Spiegel S (1998) Glycosphingolipid composition of MDA-MB-231 and MCF-7 human breast cancer cell lines. Breast Cancer Res Treat 48:149–157

    Article  PubMed  CAS  Google Scholar 

  • Nojiri H, Manya H, Isono H, Yamana H, Nojima S (1999) Induction of terminal differentiation and apoptosis in human colonic carcinoma cells by brefeldin A, a drug affecting ganglioside biosynthesis. FEBS Lett 453:140–144

    Article  PubMed  CAS  Google Scholar 

  • Nonnenmacher TF, Baumann G, Barth A, Losa GA (1994) Digital image analysis of self-similar cell profiles. J Biomed Comp 37:131–138

    Article  CAS  Google Scholar 

  • Oberhammer F, Fritsch G, Schmied M, Pavelka M, Printz D, Purchio T, Lassmann H, Schulte-Hermann R (1993) Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease. J Cellular Sci 104:317–326

    CAS  Google Scholar 

  • Ormerod MG, Collins MKL, Rodriguez-Tarduchy G, Robertson D (1992) Apoptosis in interleukin-3-dependent haemopoietic cells. J Immunol Methods 153:56–57

    Article  Google Scholar 

  • Paumgartner D, Losa GA, Weibel ER (1981) Resolution effect on the stereological estimation of surface and volume and its interpretation in terms of fractal dimensions. J Microsc 121:51–63

    PubMed  CAS  Google Scholar 

  • Pellicciari C, Bottone MG, Schaack V, Barni S, Manfredi AA (1996) Spontaneous apoptosis of thymocytes is uncoupled with progression through the cell cycle. Exp Cell Res 229:370–377

    Article  PubMed  CAS  Google Scholar 

  • Piantanelli A, Serresi S, Ricotti G, Rossolini G, Zaia A, Basso A, Piantanelli L (2002) Color based method for fractal dimension estimation of pigmented skin lesion contour. In: Losa GA, Merlini D, Nonnenmacher TF, Weibel ER (eds) Fractals in biology and medicine, vol 3. Birkhäuser, Basel Boston Berlin, pp 127–136

    Google Scholar 

  • Pican N, Trucco E, Ross M, Lane DM, Petillot Y, Ruiz IT (1998) Texture analysis for seabed classification: co-occurrence matrices versus self-organizing maps. IEEE Trans Biomed Eng 7:424–428

    Google Scholar 

  • Raff M (1998) Cell suicide for beginners. Nature 396:119–122

    Article  PubMed  CAS  Google Scholar 

  • Rigaut JP (1987) Natural objects show fractal grey tone functions: a novel approach to automated image segmentation, using mathematical morphology. Acta Stereol 6:799–810

    Google Scholar 

  • Roy HK, Iversen P, Hart J, Liu Y, Koetsier L, Kim Y, Kunte DP, Madugula M, Backman V, Wali RK (2004) Down-regulation of SNAIL suppresses MIN mouse tumorigenesis: modulation of apoptosis, proliferation, and fractal dimension. Mol Cancer Ther 3:1159–1165

    Article  PubMed  CAS  Google Scholar 

  • Santoro R, Marinelli F, Turchetti G, Zini N, Scotlandi K, Falcieri R, Maraldi NM (2002) Fractal analysis of chromatin during apoptosis. In: Losa GA, Merlini D, Nonnenmacher TF, Weibel ER (eds) Fractals in biology and medicine, vol 3. Birkhäuser, Basel Boston Berlin, pp 77–84

    Google Scholar 

  • Savill JS (1997) Recognition and phagocytosis of cells undergoing apoptosis. Br Med Bull 53:491–508

    PubMed  CAS  Google Scholar 

  • Schwartzman RA, Cidlowski JA (1993) Apoptosis: the biochemistry and molecolar biology of programmed cell death. Endocr Rev 14:133–151

    Article  PubMed  CAS  Google Scholar 

  • Sepulcre F, Grau J, Garcia-Bonafé M, Moragas A (1998) Fractal approach to the study of chromatin texture. In: Losa GA, Merlini D, Nonnenmacher TF, Weibel ER (eds) Fractals in biology and medicine, vol 2. Birkhäuser, Basel Boston Berlin, pp 226–230

    Google Scholar 

  • Spiegel S, Merrill AHJ (1996) Sphingolipid metabolism and cell growth regulation. FASEB J 10:1388–1397

    PubMed  CAS  Google Scholar 

  • Squier MKT, Cohen JJ (1997) Calpain, an upstream regulator of thymocyte apoptosis. J Immunol 158:3690–3697

    PubMed  CAS  Google Scholar 

  • Stuppia L, Gobbi P, Zamai L, Vitale M, Falcieri E (1996) Morphometric and functional study of apoptotic cell chromatin. Cell Death Differ 3:397–405

    PubMed  CAS  Google Scholar 

  • Tuceryan M, Jain AK (1998) Texture analysis. In: Chen CH, Pau LF, Whang PSP (eds) The handbook of pattern recognition and computer vision. World Scientific Publishing, Singapore, pp 207–248

    Google Scholar 

  • Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluoroscein labelled annexin V. J Immunol Methods 184:39–51

    Article  PubMed  CAS  Google Scholar 

  • Wagener R, Kobbe B, Stoffel W (1996) Quantification of gangliosides by microbore high performance liquid chromatography. J Lipid Res 37:1823–1829

    PubMed  CAS  Google Scholar 

  • Wehn B, Wouwer GVD, Koprowski M, Daele AV, Dhaene K, Scheunders P, Jakob W, Marck EV (1999) Value of morphometry, texture analysis, densitometry, and histometry in the differential diagnosis and prognosis of malignant mesothelioma. J Pathol 189:581–589

    Article  PubMed  Google Scholar 

  • Wyllie AH, Kerr JFR, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cyt 68:251–306

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Helpful discussions with Prof. Theo Nonnenmacher, University of Ulm, Germany and Prof. Carlo Pellicciari, University of Pavia, Italy are gratefully acknowledged.

Author information

Author notes

  1. Christian Castelli

    Present address: OCL Hospital, 6900, Lugano, Switzerland

Authors and Affiliations

  1. Institute for Scientific Interdisciplinary Studies, Via F. Rusca 1, P.O. Box 1132, 6600, Locarno, Switzerland

    Gabriele A. Losa & Christian Castelli

  2. Faculty of Biology and Medicine, University of Lausanne, 1015, Lausanne, Switzerland

    Gabriele A. Losa

Authors
  1. Gabriele A. Losa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christian Castelli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gabriele A. Losa.

Additional information

This work was supported by grant 31-57626.99 from the Swiss National Science Foundation and grant FOR 450.94 from the Swiss Cancer League.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Losa, G.A., Castelli, C. Nuclear patterns of human breast cancer cells during apoptosis: characterisation by fractal dimension and co-occurrence matrix statistics. Cell Tissue Res 322, 257–267 (2005). https://doi.org/10.1007/s00441-005-0030-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-005-0030-2

Keywords

Search

Navigation