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Mammary carcinoma behavior is programmed in the precancer stem cell.

Damonte P, Hodgson JG, Chen JQ, Young LJ, Cardiff RD, Borowsky AD - Breast Cancer Res. (2008)

Bottom Line: No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally.Telomerase activity is increased in both the MINO and the derived tumors when compared with normal mouse mammary gland.Within the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic 'hits' for transformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Center for Comparative Medicine, UC Davis, County Road 98 and Hutchison Drive, Davis, California 95616, USA.

ABSTRACT

Introduction: The 'MINO' (mammary intraepithelial neoplasia outgrowth) mouse model of ductal carcinoma in situ (DCIS) consists of six lines with distinct morphologic phenotypes and behavior, each meeting experimentally defined criteria for 'precancer'. Specifically, these lines grow orthotopically in cleared mammary fat pads and consistently progress to an invasive phenotype that is capable of ectopic growth. Transition to carcinoma has a consistent latency for each line, and three of the lines also exhibit pulmonary metastatic potential.

Methods: Gland cleared orthotopic transplanted precancer MINO tissues were analyzed by bacterial artifical chromosome and oligo array comparative genomic hybridization, microsatellite PCR, and telomerase repeat amplification assay. MINO cells were dissociated and cultured in three dimensional culture and transplanted in syngeneic gland cleared mammary fat pads.

Results: Comparative genomic hybridization shows that the precancer and invasive tumors are genetically stable, with low level changes including whole chromosome gains in some lines. No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally. Microsatellite analysis shows a low frequency of alterations that are predominantly permanent within a MINO line. Telomerase activity is increased in both the MINO and the derived tumors when compared with normal mouse mammary gland. Dissociation of the precancer lesion cells and three dimensional 'spheroid' culture of single cells reveals a bipotential for myoepithelial and luminal differentiation and the formation of unique three-dimensional 'MINOspheres'. These MINOspheres exhibit features that are intermediate between spheroids that are derived from normal and carcinoma cells. Transplantation of a single cell derived MINOsphere recapitulates the outgrowth of the precancer morphology and progression to carcinoma.

Conclusion: These data establish a precancer 'stem' cell that is capable of self-renewal and multilineage differentiation as the origin of invasive cancer. Within the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic 'hits' for transformation.

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Telomerase activity of MINO and tumor tissues. Telomerase activity by telomerase repeat amplification protocol (TRAP) assay. Representative ethidium stained polyacrylamide gel electrophoresis of the PCR products resulting from protein extract incubation with artificial telomere repeat template showing a bright 50 bp band with 6 bp increasing length ladder. At the bottom of the gel is a 36 bp internal PCR control, with PCR optimized to be semi-competitive with the extended telomere repeats produced by the telomerase protein derived from each sample. The protein samples are diluted in buffer and then added to the reaction mixture to provide a final reaction mixture concentration as listed (top of each lane). Matched MINO and tumor samples were tested. (a) Lines 4w4 and 4w11 are shown, with normal mouse mammary epithelium control (separated from the stroma by partial tissue dissociation and centrifugation) shown in the right three lanes. (b) Quantitation by comparing the band intensity with the internal control for multiple samples is shown, with standard deviation of the mean depicted for each bar. Line 11 tumor had the highest levels, at a mean of 500 (not shown), and all samples except for line B MINO were statistically significantly different from the normal control. There was a high level of variability between assay runs, depicted by the size of the error bars, but the qualitative data were clear, as shown in the gel (panel a). bp, base pairs; MINO, mammary intraepithelial neoplasia outgrowth; TRAP, telomerase repeat amplification protocol.
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Figure 2: Telomerase activity of MINO and tumor tissues. Telomerase activity by telomerase repeat amplification protocol (TRAP) assay. Representative ethidium stained polyacrylamide gel electrophoresis of the PCR products resulting from protein extract incubation with artificial telomere repeat template showing a bright 50 bp band with 6 bp increasing length ladder. At the bottom of the gel is a 36 bp internal PCR control, with PCR optimized to be semi-competitive with the extended telomere repeats produced by the telomerase protein derived from each sample. The protein samples are diluted in buffer and then added to the reaction mixture to provide a final reaction mixture concentration as listed (top of each lane). Matched MINO and tumor samples were tested. (a) Lines 4w4 and 4w11 are shown, with normal mouse mammary epithelium control (separated from the stroma by partial tissue dissociation and centrifugation) shown in the right three lanes. (b) Quantitation by comparing the band intensity with the internal control for multiple samples is shown, with standard deviation of the mean depicted for each bar. Line 11 tumor had the highest levels, at a mean of 500 (not shown), and all samples except for line B MINO were statistically significantly different from the normal control. There was a high level of variability between assay runs, depicted by the size of the error bars, but the qualitative data were clear, as shown in the gel (panel a). bp, base pairs; MINO, mammary intraepithelial neoplasia outgrowth; TRAP, telomerase repeat amplification protocol.

Mentions: In order to evaluate the activity of telomerase in the MINO tissues and matched tumors as compared with normal mammary epithelium, we performed the telomerase repeat amplification protocol (TRAP) assay. A sample of the polyacrylamide gel electrophoresis is shown in Figure 2a. Here the TRAP assay showed the formation of an extended ladder of six base repeats but with a quantitative competition between the telomerase repeat template and the internal control (36 bp) for PCR. The ratio of band intensity (50 bp:36 bp) was used for quantification. The average for this ratio divided by the protein concentration was recorded for each sample. The resulting representation of quantitative data is shown in Figure 2b. This histogram shows a consistently higher telomerase activity in MINO and tumor tissues compared with normal tissue. Significant variability between samples was observed. Some lanes were considered 'failed reactions' and were discarded. The variability contributes to the size of the error bars, but in all cases, except for the line B MINO, the error does not overlap with normal. The data also present a higher average telomerase activity in tumors relative to the MINOs in which they arose, but this is not statistically significant. This trend may be related to an increased contribution from normal stroma in the MINO tissue compared with the tumor tissue samples. In many studies the subjective analysis of ladder intensity and height (repeat length) was more clearly increased from normal than indicated by the quantitative ratio, but this subjective analysis (as illustrated in Figure 2a) was not quantified. Notably, consistent with the literature, the normal mouse mammary gland has significantly higher telomerase activity than normal human tissues.


Mammary carcinoma behavior is programmed in the precancer stem cell.

Damonte P, Hodgson JG, Chen JQ, Young LJ, Cardiff RD, Borowsky AD - Breast Cancer Res. (2008)

Telomerase activity of MINO and tumor tissues. Telomerase activity by telomerase repeat amplification protocol (TRAP) assay. Representative ethidium stained polyacrylamide gel electrophoresis of the PCR products resulting from protein extract incubation with artificial telomere repeat template showing a bright 50 bp band with 6 bp increasing length ladder. At the bottom of the gel is a 36 bp internal PCR control, with PCR optimized to be semi-competitive with the extended telomere repeats produced by the telomerase protein derived from each sample. The protein samples are diluted in buffer and then added to the reaction mixture to provide a final reaction mixture concentration as listed (top of each lane). Matched MINO and tumor samples were tested. (a) Lines 4w4 and 4w11 are shown, with normal mouse mammary epithelium control (separated from the stroma by partial tissue dissociation and centrifugation) shown in the right three lanes. (b) Quantitation by comparing the band intensity with the internal control for multiple samples is shown, with standard deviation of the mean depicted for each bar. Line 11 tumor had the highest levels, at a mean of 500 (not shown), and all samples except for line B MINO were statistically significantly different from the normal control. There was a high level of variability between assay runs, depicted by the size of the error bars, but the qualitative data were clear, as shown in the gel (panel a). bp, base pairs; MINO, mammary intraepithelial neoplasia outgrowth; TRAP, telomerase repeat amplification protocol.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2481504&req=5

Figure 2: Telomerase activity of MINO and tumor tissues. Telomerase activity by telomerase repeat amplification protocol (TRAP) assay. Representative ethidium stained polyacrylamide gel electrophoresis of the PCR products resulting from protein extract incubation with artificial telomere repeat template showing a bright 50 bp band with 6 bp increasing length ladder. At the bottom of the gel is a 36 bp internal PCR control, with PCR optimized to be semi-competitive with the extended telomere repeats produced by the telomerase protein derived from each sample. The protein samples are diluted in buffer and then added to the reaction mixture to provide a final reaction mixture concentration as listed (top of each lane). Matched MINO and tumor samples were tested. (a) Lines 4w4 and 4w11 are shown, with normal mouse mammary epithelium control (separated from the stroma by partial tissue dissociation and centrifugation) shown in the right three lanes. (b) Quantitation by comparing the band intensity with the internal control for multiple samples is shown, with standard deviation of the mean depicted for each bar. Line 11 tumor had the highest levels, at a mean of 500 (not shown), and all samples except for line B MINO were statistically significantly different from the normal control. There was a high level of variability between assay runs, depicted by the size of the error bars, but the qualitative data were clear, as shown in the gel (panel a). bp, base pairs; MINO, mammary intraepithelial neoplasia outgrowth; TRAP, telomerase repeat amplification protocol.
Mentions: In order to evaluate the activity of telomerase in the MINO tissues and matched tumors as compared with normal mammary epithelium, we performed the telomerase repeat amplification protocol (TRAP) assay. A sample of the polyacrylamide gel electrophoresis is shown in Figure 2a. Here the TRAP assay showed the formation of an extended ladder of six base repeats but with a quantitative competition between the telomerase repeat template and the internal control (36 bp) for PCR. The ratio of band intensity (50 bp:36 bp) was used for quantification. The average for this ratio divided by the protein concentration was recorded for each sample. The resulting representation of quantitative data is shown in Figure 2b. This histogram shows a consistently higher telomerase activity in MINO and tumor tissues compared with normal tissue. Significant variability between samples was observed. Some lanes were considered 'failed reactions' and were discarded. The variability contributes to the size of the error bars, but in all cases, except for the line B MINO, the error does not overlap with normal. The data also present a higher average telomerase activity in tumors relative to the MINOs in which they arose, but this is not statistically significant. This trend may be related to an increased contribution from normal stroma in the MINO tissue compared with the tumor tissue samples. In many studies the subjective analysis of ladder intensity and height (repeat length) was more clearly increased from normal than indicated by the quantitative ratio, but this subjective analysis (as illustrated in Figure 2a) was not quantified. Notably, consistent with the literature, the normal mouse mammary gland has significantly higher telomerase activity than normal human tissues.

Bottom Line: No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally.Telomerase activity is increased in both the MINO and the derived tumors when compared with normal mouse mammary gland.Within the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic 'hits' for transformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Center for Comparative Medicine, UC Davis, County Road 98 and Hutchison Drive, Davis, California 95616, USA.

ABSTRACT

Introduction: The 'MINO' (mammary intraepithelial neoplasia outgrowth) mouse model of ductal carcinoma in situ (DCIS) consists of six lines with distinct morphologic phenotypes and behavior, each meeting experimentally defined criteria for 'precancer'. Specifically, these lines grow orthotopically in cleared mammary fat pads and consistently progress to an invasive phenotype that is capable of ectopic growth. Transition to carcinoma has a consistent latency for each line, and three of the lines also exhibit pulmonary metastatic potential.

Methods: Gland cleared orthotopic transplanted precancer MINO tissues were analyzed by bacterial artifical chromosome and oligo array comparative genomic hybridization, microsatellite PCR, and telomerase repeat amplification assay. MINO cells were dissociated and cultured in three dimensional culture and transplanted in syngeneic gland cleared mammary fat pads.

Results: Comparative genomic hybridization shows that the precancer and invasive tumors are genetically stable, with low level changes including whole chromosome gains in some lines. No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally. Microsatellite analysis shows a low frequency of alterations that are predominantly permanent within a MINO line. Telomerase activity is increased in both the MINO and the derived tumors when compared with normal mouse mammary gland. Dissociation of the precancer lesion cells and three dimensional 'spheroid' culture of single cells reveals a bipotential for myoepithelial and luminal differentiation and the formation of unique three-dimensional 'MINOspheres'. These MINOspheres exhibit features that are intermediate between spheroids that are derived from normal and carcinoma cells. Transplantation of a single cell derived MINOsphere recapitulates the outgrowth of the precancer morphology and progression to carcinoma.

Conclusion: These data establish a precancer 'stem' cell that is capable of self-renewal and multilineage differentiation as the origin of invasive cancer. Within the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic 'hits' for transformation.

Show MeSH
Related in: MedlinePlus