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.
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.
Dalerba
Piero
P
Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Milan, Italy.
Guiducci
Cristiana
C
Poliani
Pietro Luigi
PL
Cifola
Ingrid
I
Parenza
Mariella
M
Frattini
Milo
M
Gallino
Gianfrancesco
G
Carnevali
Ileana
I
Di Giulio
Ilvia
I
Andreola
Salvatore
S
Lombardo
Claudia
C
Rivoltini
Licia
L
Schweighoffer
Tamás
T
Belli
Filiberto
F
Colombo
Mario P
MP
Parmiani
Giorgio
G
Castelli
Chiara
C
D000818:Animals; D016922:Cellular Senescence / Q000502:physiology; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D004268:DNA-Binding Proteins; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D019098:Telomerase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D016615:Telomere / Q000502:physiology; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D019098:Telomerase