Multipotent human breast stem cell line MCF1OAT

Tait, Larry; Dawson, Peter Paul; Wolman, Sandra R.; Galea, Karen S.; Miller, Fred R.

doi:10.3892/ijo.9.2.263

Cited by 9 publications

(9 citation statements)

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“…H1299, the breast carcinoma cell lines MPE600 and ZR75-30, the uveal melanoma cell lines MEL285 and 92.1 (38), N-TERA-2, 833KE, and mouse melanoma B16F10 cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum. The generation and culture conditions of MCF-10A (M1) and MCF-10AT (M2) cells have been described (39). To generate stable p53 knockdown and control knockdown cell lines, cells were infected with lentiviral vectors expressing shRNA targeting human p53 or mouse mdmx and conferring puromycin resistance.…”

Section: Methodsmentioning

confidence: 99%

HDMX-L Is Expressed from a Functional p53-responsive Promoter in the First Intron of the HDMX Gene and Participates in an Autoregulatory Feedback Loop to Control p53 Activity

Phillips

Teunisse

Lam

et al. 2010

Journal of Biological Chemistry

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The p53 regulatory network is critically involved in preventing the initiation of cancer. In unstressed cells, p53 is maintained at low levels and is largely inactive, mainly through the action of its two essential negative regulators, HDM2 and HDMX. p53 abundance and activity are up-regulated in response to various stresses, including DNA damage and oncogene activation. Active p53 initiates transcriptional and transcription-independent programs that result in cell cycle arrest, cellular senescence, or apoptosis. p53 also activates transcription of HDM2, which initially leads to the degradation of HDMX, creating a positive feedback loop to obtain maximal activation of p53. Subsequently, when stress-induced post-translational modifications start to decline, HDM2 becomes effective in targeting p53 for degradation, thus attenuating the p53 response. To date, no clear function for HDMX in this critical attenuation phase has been demonstrated experimentally. Like HDM2, the HDMX gene contains a promoter (P2) in its first intron that is potentially inducible by p53. We show that p53 activation in response to a plethora of p53-activating agents induces the transcription of a novel HDMX mRNA transcript from the HDMX-P2 promoter. This mRNA is more efficiently translated than that expressed from the constitutive HDMX-P1 promoter, and it encodes a long form of HDMX protein, HDMX-L. Importantly, we demonstrate that HDMX-L cooperates with HDM2 to promote the ubiquitination of p53 and that p53-induced HDMX transcription from the P2 promoter can play a key role in the attenuation phase of the p53 response, to effectively diminish p53 abundance as cells recover from stress.The tumor suppressor protein p53 functions primarily as a stress-inducible transcriptional activator of genes that promote cell cycle arrest and apoptosis (1). Stress-induced p53 activation can form a rate-limiting barrier to tumorigenesis (2, 3), and the manipulation of p53 function is key to the mechanism of action of many cancer chemotherapeutic strategies (4, 5). In unstressed cells, p53 is maintained at low levels and inactive, largely through the action of several p53-inducible negative feedback pathways, the most extensively studied of which involves the oncoproteins HDM2 and HDMX (also called MDM4) (MDM2 and MDMX/MDM4 in mice) (6, 7). Considerable research effort has been applied to understanding the mechanisms whereby these two proteins regulate p53 function. HDM2 and HDMX both contain an N-terminal pocket that binds to the primary transactivation domain of p53; they can, therefore, function independently of each other to repress p53-dependent transcription (8 -10). HDM2 also forms both HDM2-HDM2 homodimers and HDM2-HDMX heterodimers. These function as E3 ubiquitin ligases for p53; monoubiquitination of p53 by HDM2 inhibits p53 activity by both inhibiting acetylation and promoting nuclear export, whereas polyubiquitination promotes proteasome-mediated p53 degradation and is largely responsible for the rapid turnover of p53 protein that occurs in proli...

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Section: Methodsmentioning

confidence: 99%

HDMX-L Is Expressed from a Functional p53-responsive Promoter in the First Intron of the HDMX Gene and Participates in an Autoregulatory Feedback Loop to Control p53 Activity

Phillips

Teunisse

Lam

et al. 2010

Journal of Biological Chemistry

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“…The presence of basement membrane was demonstrated by staining with silver methamine-borate (Sigma Diagnostics) as described previously. 36 In some cases, sections were dual stained with α-smooth muscle actin antibody and silver stain for localization of myoepithelium and basement membrane.…”

Section: Methodsmentioning

confidence: 99%

Comedo-Ductal carcinoma in situ: A paradoxical role for programmed cell death

Shekhar¹,

Tait²,

Pauley³

et al. 2008

Cancer Biology & Therapy

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Comedo-DCIS is a histologic subtype of preinvasive breast neoplasia that is characterized by prominent apoptotic cell death and has greater malignant potential than other DCIS subtypes. We investigated the mechanisms of apoptosis in comedo-DCIS and its role in conversion of comedo-DCIS to invasive cancer. Clinical comedo-DCIS excisions and the MCF10DCIS.com human breast cancer model which produces lesions resembling comedo-DCIS were analyzed. Apoptotic luminal and myoepithelial cells were identified by TUNEL and reactivity to cleaved PARP antibody and cell death assessed by Western blotting, Mitocapture and immunohistochemical assays. MCF10DCIS.com cells undergo spontaneous apoptosis in vitro, both in monolayers and multicellular spheroids; it is associated with increased mitochondrial membrane permeability, increase in Bax/Bcl-2 ratio and occurs via caspase-9-dependent p53-independent pathway. This suggests that apoptosis is stromal-independent and that the cells are programmed to undergo apoptosis. Immunostaining with cleaved PARP antibody showed that myoepithelial apoptosis occurs before lesions progress to comedo-DCIS in both clinical comedo-DCIS and in vivo MCF10DCIS.com lesions. Intense staining for MMP-2, MMP-3, MMP-9 and MMP-11 was observed in the stroma and epithelia of solid DCIS lesions prior to conversion to comedo-DCIS in clinical and MCF10DCIS.com lesions. Gelatin zymography showed higher MMP-2 levels in lysates and conditioned media of MCF10DCIS.com cells undergoing apoptosis. These data suggest that signals arising from the outside (microenvironmental) and inside (internal genetic alterations) of the duct act in concert to trigger apoptosis of myoepithelial and luminal epithelial cells. Our findings implicate spontaneous apoptosis in both the etiology and progression of comedo-DCIS. It is possible that spontaneous apoptosis facilitates elimination of cells thus permitting expansion and malignant transformation of cancer cells that are resistant to spontaneous apoptosis.

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“…These cells were derived from the immortalized MCF-10A cells via transformation with T24 mutant c-Ha-ras (27,28). Interestingly, the MCF10AT cells appear to contain multipotent (or bipotential) breast stem cells because both luminal epithelial and myoepithelial cells can be derived from these cells in vivo (29). A derivative of the MCF10AT premalignant human cell line model was established MCF10DCIS.com that reproducibly forms comedo DCIS-like lesions that spontaneously progress to invasive tumors (27,28).…”

Section: Experimental Models Of Human Dcismentioning

confidence: 99%

Molecular Markers for the Diagnosis and Management of Ductal Carcinoma In Situ

Polyák

2010

JNCI Monographs

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Cancer is an evolution of a population of cancer cells with diverse hereditary characteristics. With rare exceptions, tumors are derived from a single initiated cell and the progressive accumulation of genetic, epigenetic, and phenotypic features combined with selection drives cancer progression. The currently accepted stepwise model of breast tumorigenesis assumes a gradual transition from epithelial hyperproliferation to ductal carcinoma in situ (DCIS), to invasive and metastatic carcinomas (1,2). This progression model is strongly supported by human clinical and epidemiological data and by molecular clonality studies addressing the relationships between in situ and invasive areas of the same tumor and between DCIS and its local invasive recurrence. Until 1980, DCIS was diagnosed very rarely and represented less than 1% of all breast cancer cases. All of this changed dramatically with the increased utilization of mammography during the 1980s, and DCIS became the most rapidly increasing subset of breast cancers. Currently, DCIS accounts for 15%-25% of newly diagnosed breast cancer cases in the United States (3). In contrast to the dramatic improvement in our ability to detect DCIS, our understanding of the pathophysiology of this disease and factors involved in its progression to invasive carcinoma are still poorly defined. Numerous studies compared the gene expression, genetic, and epigenetic profiles of DCIS and invasive breast carcinomas, but a molecular alteration differentiating in situ and invasive tumors has not been identified (2). A dramatic change occurs during the normal to DCIS transition, but surprisingly in situ and invasive breast carcinomas of the same histological subtype essentially share the same genetic and epigenetic alterations and expression patterns. In contrast, the molecular profiles of breast tumors of distinct subtypes (ie, luminal, HER2+, and basal-like) are dramatically different. Emerging data also suggest a critical role for microenvironmental changes in the regulation of tumor progression, particularly that of the in situ to invasive breast cancer transition (4). Here, I summarize the results of molecular studies aiming to identify diagnostic markers that differentiate in situ and invasive tumors and predictive markers that correlate with the risk of invasive progression. Candidate Molecular Markers in DCISThe expression and mutation status of numerous tumor suppressor and oncogenes have been analyzed in DCIS and invasive ductal carcinoma (IDC) including TP53, PTEN, PIK3CA, ERBB2, MYC, and differences in the frequency of these changes have been found according to the tumor subtype but not histological stage (2). Thus, mutations in TP53 are more frequent in basal-like and HER2+ compared with luminal tumors; in basal-like cases, PIK3CA is rarely mutated but PTEN is frequently lost, and amplification of ERBB2 is specific for the HER2+ subtype.The expression of several candidate genes selected based on their biological function has also been analyzed in DCIS (5). Two recent studies ...

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Multipotent human breast stem cell line MCF1OAT

Cited by 9 publications

References 0 publications

HDMX-L Is Expressed from a Functional p53-responsive Promoter in the First Intron of the HDMX Gene and Participates in an Autoregulatory Feedback Loop to Control p53 Activity

HDMX-L Is Expressed from a Functional p53-responsive Promoter in the First Intron of the HDMX Gene and Participates in an Autoregulatory Feedback Loop to Control p53 Activity

Comedo-Ductal carcinoma in situ: A paradoxical role for programmed cell death

Molecular Markers for the Diagnosis and Management of Ductal Carcinoma In Situ

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