Reconstitution of human telomerase reverse transcriptase expression rescues colorectal carcinoma cells from in vitro senescence

Evidence against immortality as a constitutive trait of tumor cells

Piero Dalerba, Cristiana Guiducci, Pietro Luigi Poliani, Ingrid Cifola, Mariella Parenza, Milo Frattini, Gianfrancesco Gallino, Ileana Carnevali, Ilvia Di Giulio, Salvatore Andreola, Claudia Lombardo, Licia Rivoltini, Tamás Schweighoffer, Filiberto Belli, Mario P. Colombo, Giorgio Parmiani, Chiara Castelli

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Although in vitro establishment of new colorectal carcinoma (CRC) cell lines is an infrequent event, we have observed that primary cultures of CRC can be repeatedly and reproducibly initiated following in vitro plating of tumor-derived epithelial cells. These cultures, however, usually display a short life span as they undergo a limited number of cell passages before entering a state of irreversible growth arrest. In this study, we show that short-lived CRC primary cultures lack constitutive telomerase activity and undergo a senescence process characterized by progressive telomere shortening. Moreover, transduction of these cells with a retroviral vector encoding human telomerase reverse transcriptase (hTERT) is sufficient to reconstitute telomerase activity and allow immortalization. Detailed molecular characterization of hTERT-immortalized CRC cell lines confirms their individual tumor origin by showing expression of colonic epithelial differentiation markers, such as cytokeratin-20 (CK20), full match with class I and class II human leukocyte antigen genotyping of autologous B-lymphoblastoid cells, and presence of somatic mutations in key cancer genes (KRAS2, APC) identical to those of the corresponding autologous original tumor tissues. Moreover, functional characterization of hTERT-immortalized CRC cell lines shows that they have a transformed phenotype, being able to form colonies in soft agar and tumors in severe combined immunodeficient mice. Most interestingly, immunohistochemical analysis of original tumor tissues indicates that short-lived CRC primary cultures, although hTERT-negative in vitro, derive from hTERT-positive tumors. Taken together, our data show that, in a least subset of CRC, biochemical pathways involved in maintenance of telomere length, such as telomerase, are not activated in a constitutive way in all tumor cells.

Original languageEnglish
Pages (from-to)2321-2329
Number of pages9
JournalCancer Research
Volume65
Issue number6
DOIs
Publication statusPublished - Mar 15 2005

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Colorectal Neoplasms
Telomerase
Neoplasms
Cell Line
Telomere Homeostasis
Keratin-20
HLA-D Antigens
Telomere Shortening
SCID Mice
Neoplasm Genes
Differentiation Antigens
HLA Antigens
In Vitro Techniques
human TERT protein
Agar
Cell Count
Epithelial Cells
Phenotype
Mutation
Growth

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Reconstitution of human telomerase reverse transcriptase expression rescues colorectal carcinoma cells from in vitro senescence : Evidence against immortality as a constitutive trait of tumor cells. / Dalerba, Piero; Guiducci, Cristiana; Poliani, Pietro Luigi; Cifola, Ingrid; Parenza, Mariella; Frattini, Milo; Gallino, Gianfrancesco; Carnevali, Ileana; Di Giulio, Ilvia; Andreola, Salvatore; Lombardo, Claudia; Rivoltini, Licia; Schweighoffer, Tamás; Belli, Filiberto; Colombo, Mario P.; Parmiani, Giorgio; Castelli, Chiara.

In: Cancer Research, Vol. 65, No. 6, 15.03.2005, p. 2321-2329.

Research output: Contribution to journalArticle

Dalerba, Piero ; Guiducci, Cristiana ; Poliani, Pietro Luigi ; Cifola, Ingrid ; Parenza, Mariella ; Frattini, Milo ; Gallino, Gianfrancesco ; Carnevali, Ileana ; Di Giulio, Ilvia ; Andreola, Salvatore ; Lombardo, Claudia ; Rivoltini, Licia ; Schweighoffer, Tamás ; Belli, Filiberto ; Colombo, Mario P. ; Parmiani, Giorgio ; Castelli, Chiara. / Reconstitution of human telomerase reverse transcriptase expression rescues colorectal carcinoma cells from in vitro senescence : Evidence against immortality as a constitutive trait of tumor cells. In: Cancer Research. 2005 ; Vol. 65, No. 6. pp. 2321-2329.
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abstract = "Although in vitro establishment of new colorectal carcinoma (CRC) cell lines is an infrequent event, we have observed that primary cultures of CRC can be repeatedly and reproducibly initiated following in vitro plating of tumor-derived epithelial cells. These cultures, however, usually display a short life span as they undergo a limited number of cell passages before entering a state of irreversible growth arrest. In this study, we show that short-lived CRC primary cultures lack constitutive telomerase activity and undergo a senescence process characterized by progressive telomere shortening. Moreover, transduction of these cells with a retroviral vector encoding human telomerase reverse transcriptase (hTERT) is sufficient to reconstitute telomerase activity and allow immortalization. Detailed molecular characterization of hTERT-immortalized CRC cell lines confirms their individual tumor origin by showing expression of colonic epithelial differentiation markers, such as cytokeratin-20 (CK20), full match with class I and class II human leukocyte antigen genotyping of autologous B-lymphoblastoid cells, and presence of somatic mutations in key cancer genes (KRAS2, APC) identical to those of the corresponding autologous original tumor tissues. Moreover, functional characterization of hTERT-immortalized CRC cell lines shows that they have a transformed phenotype, being able to form colonies in soft agar and tumors in severe combined immunodeficient mice. Most interestingly, immunohistochemical analysis of original tumor tissues indicates that short-lived CRC primary cultures, although hTERT-negative in vitro, derive from hTERT-positive tumors. Taken together, our data show that, in a least subset of CRC, biochemical pathways involved in maintenance of telomere length, such as telomerase, are not activated in a constitutive way in all tumor cells.",
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AU - Poliani, Pietro Luigi

AU - Cifola, Ingrid

AU - Parenza, Mariella

AU - Frattini, Milo

AU - Gallino, Gianfrancesco

AU - Carnevali, Ileana

AU - Di Giulio, Ilvia

AU - Andreola, Salvatore

AU - Lombardo, Claudia

AU - Rivoltini, Licia

AU - Schweighoffer, Tamás

AU - Belli, Filiberto

AU - Colombo, Mario P.

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AU - Castelli, Chiara

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N2 - Although in vitro establishment of new colorectal carcinoma (CRC) cell lines is an infrequent event, we have observed that primary cultures of CRC can be repeatedly and reproducibly initiated following in vitro plating of tumor-derived epithelial cells. These cultures, however, usually display a short life span as they undergo a limited number of cell passages before entering a state of irreversible growth arrest. In this study, we show that short-lived CRC primary cultures lack constitutive telomerase activity and undergo a senescence process characterized by progressive telomere shortening. Moreover, transduction of these cells with a retroviral vector encoding human telomerase reverse transcriptase (hTERT) is sufficient to reconstitute telomerase activity and allow immortalization. Detailed molecular characterization of hTERT-immortalized CRC cell lines confirms their individual tumor origin by showing expression of colonic epithelial differentiation markers, such as cytokeratin-20 (CK20), full match with class I and class II human leukocyte antigen genotyping of autologous B-lymphoblastoid cells, and presence of somatic mutations in key cancer genes (KRAS2, APC) identical to those of the corresponding autologous original tumor tissues. Moreover, functional characterization of hTERT-immortalized CRC cell lines shows that they have a transformed phenotype, being able to form colonies in soft agar and tumors in severe combined immunodeficient mice. Most interestingly, immunohistochemical analysis of original tumor tissues indicates that short-lived CRC primary cultures, although hTERT-negative in vitro, derive from hTERT-positive tumors. Taken together, our data show that, in a least subset of CRC, biochemical pathways involved in maintenance of telomere length, such as telomerase, are not activated in a constitutive way in all tumor cells.

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