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Immortalization and transformation of human mammary epithelial cells by a tumor-derived Myc mutant.

ABSTRACT: The Myc transcription factor is commonly dysregulated in many human cancers, including breast carcinomas. However, the precise role of Myc in the initiation and maintenance of malignancy is unclear. In this study we compared the ability of wild-type Myc (wt Myc) or Myc phosphorylation deficient mutants (T58A, S62A or T58A/S62A) to immortalize and transform human mammary epithelial cells (HMECs). All Myc constructs promoted cellular immortalization. As previously reported in other cells, the Myc T58A mutant tempered apoptotic responses and increased Myc protein stability in HMEC cells. More importantly, we now show that HMECs overexpressing the Myc T58A mutant acquire a unique cellular phenotype characterized by cell aggregation, detachment from the substrate and growth in liquid suspension. Coincident with these changes, the cells become anchorage-independent for growth in agarose. Previous studies have shown that wt Myc can collaborate with hTERT in inducing HMEC anchorage-independent growth. We have verified this observation and further shown that Myc T58A was a stronger facilitator of such co-transformation. Thus, our findings indicate that differences in Myc protein phosphorylation modulate its biological activity in human breast epithelial cells and specifically that the T58A mutation can facilitate both cellular immortalization and transformation. Finally, we used the isogenic cell lines generated in this study to identify a subset of genes whose expression is greatly altered during the transition from the immortal to the anchorage-independent states.

SUBMITTER: Thibodeaux CA

PROVIDER: S-EPMC3576819 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Immortalization and transformation of human mammary epithelial cells by a tumor-derived Myc mutant.

Thibodeaux Clare A CA Liu Xuefeng X Disbrow Gary L GL Zhang Yiyu Y Rone Janice D JD Haddad Bassem R BR Schlegel Richard R

Breast cancer research and treatment 20080720 2

The Myc transcription factor is commonly dysregulated in many human cancers, including breast carcinomas. However, the precise role of Myc in the initiation and maintenance of malignancy is unclear. In this study we compared the ability of wild-type Myc (wt Myc) or Myc phosphorylation deficient mutants (T58A, S62A or T58A/S62A) to immortalize and transform human mammary epithelial cells (HMECs). All Myc constructs promoted cellular immortalization. As previously reported in other cells, the Myc ...[more]

PMID: 18642118

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Project description:Human neural stem cells (hNSC) represent an essential source of renewable brain cells for both experimental studies and cell replacement therapies. Their relatively slow rate of proliferation and physiological senescence in culture make their use cumbersome under some experimental and pre-clinical settings. The immortalization of hNSC with the v-myc gene (v-IhNSC) has been shown to generate stem cells endowed with enhanced proliferative capacity, which greatly facilitates the study of hNSCs, both in vitro and in vivo. Despite the excellent safety properties displayed by v-IhNSCs--which do not transform in vitro and are not tumorigenic in vivo--the v-myc gene contains several mutations and recombination elements, whose role(s) and effects remains to be elucidated, yielding unresolved safety concerns. To address this issue, we used a c-myc T58A retroviral vector to establish an immortal cell line (T-IhNSC) from the same hNSCs used to generate the original v-IhNSCs and compared their characteristics with the latter, with hNSC and with hNSC immortalized using c-myc wt (c-IhNSC). T-IhNSCs displayed an enhanced self-renewal ability, with their proliferative capacity and clonogenic potential being remarkably comparable to those of v-IhNSC and higher than wild type hNSCs and c-IhNSCs. Upon growth factors removal, T-IhNSC promptly gave rise to well-differentiated neurons, astrocytes and most importantly, to a heretofore undocumented high percentage of human oligodendrocytes (up to 23%). Persistent growth-factor dependence, steady functional properties, lack of ability to generate colonies in soft-agar colony-forming assay and to establish tumors upon orthotopic transplantation, point to the fact that immortalization by c-myc T58A does not bring about tumorigenicity in hNSCs. Hence, this work describes a novel and continuous cell line of immortalized human multipotent neural stem cells, in which the immortalizing agent is represented by a single gene which, in turn, carries a single and well characterized mutation. From a different perspective, these data report on a safe approach to increase human neural stem cells propagation in culture, without altering their basic properties. These T-IhNSC line provides a versatile model for the elucidation of the mechanisms involved in human neural stem cells expansion and for development of high throughput assays for both basic and translational research on human neural cell development. The improved proclivity of T-IhNSC to generate human oligodendrocytes propose T-IhNSC as a feasible candidate for the design of experimental and, perhaps, therapeutic approaches in demyelinating diseases.

Dataset Information

Immortalization and transformation of human mammary epithelial cells by a tumor-derived Myc mutant.

Publications

Immortalization and transformation of human mammary epithelial cells by a tumor-derived Myc mutant.

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