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Rapid spread of mouse mammary tumor virus in cultured human breast cells.

Indik S, Günzburg WH, Kulich P, Salmons B, Rouault F - Retrovirology (2007)

Bottom Line: The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody.Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine.The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Institute for Virology and Biomedicine, University of Veterinary Medicine Vienna, Vienna, A-1210, Austria. stanislav.indik@vu-wien.ac.at

ABSTRACT

Background: The role of mouse mammary tumor virus (MMTV) as a causative agent in human breast carcinogenesis has recently been the subject of renewed interest. The proposed model is based on the detection of MMTV sequences in human breast cancer but not in healthy breast tissue. One of the main drawbacks to this model, however, was that until now human cells had not been demonstrated to sustain productive MMTV infection.

Results: Here, we show for the first time the rapid spread of mouse mammary tumor virus, MMTV(GR), in cultured human mammary cells (Hs578T), ultimately leading to the infection of every cell in culture. The replication of the virus was monitored by quantitative PCR, quantitative RT-PCR and immunofluorescence imaging. The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody. Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine. The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences.

Conclusion: Taken together, our results show that human cells can support replication of mouse mammary tumor virus.

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Related in: MedlinePlus

Quantification of proviral DNA and viral RNA in cell lysates and supernatants of the third-round infected human breast cells during a time-course experiment. (A and B) The third-round infected cells were cultured in the presence (A) or absence (B) of 10-6 M DEX. Genomic DNA was extracted from the infected cells at the indicated time points and semiquantitative PCR was performed. NC: non-transduced HS578T cells. PC: second-round infected Hs578T cells. Equal DNA loading was controlled in a PCR assay with GAPDH-specific primers (bottom panels). M: 1 kb marker. (C) Real-time TaqMan PCR quantifying proviral loads in the infected Hs578T cells during the time-course experiment. (D) Equal loading of the PCR reactions was controlled in a Real-time TaqMan PCR specific for GAPDH gene. (E) The viral RNA was quantified by Real-time RT-PCR in cell culture fluids of the infected Hs578T cells grown either in the presence or absence of 10-6 M DEX.
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Figure 3: Quantification of proviral DNA and viral RNA in cell lysates and supernatants of the third-round infected human breast cells during a time-course experiment. (A and B) The third-round infected cells were cultured in the presence (A) or absence (B) of 10-6 M DEX. Genomic DNA was extracted from the infected cells at the indicated time points and semiquantitative PCR was performed. NC: non-transduced HS578T cells. PC: second-round infected Hs578T cells. Equal DNA loading was controlled in a PCR assay with GAPDH-specific primers (bottom panels). M: 1 kb marker. (C) Real-time TaqMan PCR quantifying proviral loads in the infected Hs578T cells during the time-course experiment. (D) Equal loading of the PCR reactions was controlled in a Real-time TaqMan PCR specific for GAPDH gene. (E) The viral RNA was quantified by Real-time RT-PCR in cell culture fluids of the infected Hs578T cells grown either in the presence or absence of 10-6 M DEX.

Mentions: In subsequent time-course experiments we sought to directly demonstrate the productive replication of MMTV in human breast cells. We reasoned that if human cells are capable of supporting replication of MMTV, then we should observe increasing levels of proviral DNA upon cultivation of the third-round infected cells in the presence of 10-6 M DEX, a glucocorticoid inducing MMTV expression. Indeed, as expected for an ongoing productive infection, stronger PCR signals were detected at later cultivation time points (Figure 3A). The increased intensities of the PCR signals could not be attributable to unequal loading of the amplification reaction, since a PCR assay with GAPDH-specific primers performed with the identical template amounts, showed similar levels of PCR products at all time points (Figure 3A and 3B, bottom). Furthermore, no increase in the intensity of the PCR products were detected in the cells cultured without DEX (Figure 3B). Taking this data together, we observed a time- and dexamethasone-dependent increase of MMTV-specific PCR products strongly supporting the productive infection of the human cells with MMTV. Similar results were also observed when a TaqMan Real-time PCR assay targeting the 5' end of the env coding region was used for an accurate quantification of the proviral load in the infected cells during the time-course experiment (Figure 3C). Real-time PCR assays of the GAPDH housekeeping gene confirmed equal loading of all PCR reactions (Figure 3D).


Rapid spread of mouse mammary tumor virus in cultured human breast cells.

Indik S, Günzburg WH, Kulich P, Salmons B, Rouault F - Retrovirology (2007)

Quantification of proviral DNA and viral RNA in cell lysates and supernatants of the third-round infected human breast cells during a time-course experiment. (A and B) The third-round infected cells were cultured in the presence (A) or absence (B) of 10-6 M DEX. Genomic DNA was extracted from the infected cells at the indicated time points and semiquantitative PCR was performed. NC: non-transduced HS578T cells. PC: second-round infected Hs578T cells. Equal DNA loading was controlled in a PCR assay with GAPDH-specific primers (bottom panels). M: 1 kb marker. (C) Real-time TaqMan PCR quantifying proviral loads in the infected Hs578T cells during the time-course experiment. (D) Equal loading of the PCR reactions was controlled in a Real-time TaqMan PCR specific for GAPDH gene. (E) The viral RNA was quantified by Real-time RT-PCR in cell culture fluids of the infected Hs578T cells grown either in the presence or absence of 10-6 M DEX.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2169256&req=5

Figure 3: Quantification of proviral DNA and viral RNA in cell lysates and supernatants of the third-round infected human breast cells during a time-course experiment. (A and B) The third-round infected cells were cultured in the presence (A) or absence (B) of 10-6 M DEX. Genomic DNA was extracted from the infected cells at the indicated time points and semiquantitative PCR was performed. NC: non-transduced HS578T cells. PC: second-round infected Hs578T cells. Equal DNA loading was controlled in a PCR assay with GAPDH-specific primers (bottom panels). M: 1 kb marker. (C) Real-time TaqMan PCR quantifying proviral loads in the infected Hs578T cells during the time-course experiment. (D) Equal loading of the PCR reactions was controlled in a Real-time TaqMan PCR specific for GAPDH gene. (E) The viral RNA was quantified by Real-time RT-PCR in cell culture fluids of the infected Hs578T cells grown either in the presence or absence of 10-6 M DEX.
Mentions: In subsequent time-course experiments we sought to directly demonstrate the productive replication of MMTV in human breast cells. We reasoned that if human cells are capable of supporting replication of MMTV, then we should observe increasing levels of proviral DNA upon cultivation of the third-round infected cells in the presence of 10-6 M DEX, a glucocorticoid inducing MMTV expression. Indeed, as expected for an ongoing productive infection, stronger PCR signals were detected at later cultivation time points (Figure 3A). The increased intensities of the PCR signals could not be attributable to unequal loading of the amplification reaction, since a PCR assay with GAPDH-specific primers performed with the identical template amounts, showed similar levels of PCR products at all time points (Figure 3A and 3B, bottom). Furthermore, no increase in the intensity of the PCR products were detected in the cells cultured without DEX (Figure 3B). Taking this data together, we observed a time- and dexamethasone-dependent increase of MMTV-specific PCR products strongly supporting the productive infection of the human cells with MMTV. Similar results were also observed when a TaqMan Real-time PCR assay targeting the 5' end of the env coding region was used for an accurate quantification of the proviral load in the infected cells during the time-course experiment (Figure 3C). Real-time PCR assays of the GAPDH housekeeping gene confirmed equal loading of all PCR reactions (Figure 3D).

Bottom Line: The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody.Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine.The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Institute for Virology and Biomedicine, University of Veterinary Medicine Vienna, Vienna, A-1210, Austria. stanislav.indik@vu-wien.ac.at

ABSTRACT

Background: The role of mouse mammary tumor virus (MMTV) as a causative agent in human breast carcinogenesis has recently been the subject of renewed interest. The proposed model is based on the detection of MMTV sequences in human breast cancer but not in healthy breast tissue. One of the main drawbacks to this model, however, was that until now human cells had not been demonstrated to sustain productive MMTV infection.

Results: Here, we show for the first time the rapid spread of mouse mammary tumor virus, MMTV(GR), in cultured human mammary cells (Hs578T), ultimately leading to the infection of every cell in culture. The replication of the virus was monitored by quantitative PCR, quantitative RT-PCR and immunofluorescence imaging. The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody. Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine. The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences.

Conclusion: Taken together, our results show that human cells can support replication of mouse mammary tumor virus.

Show MeSH
Related in: MedlinePlus