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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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Detection of expression of MMTV proteins in the infected human cells. (A and B) Infected Hs578T cells were cultured for 5 weeks in the presence of 10-6 M DEX. One week before immunofluorescence staining the cells were cultivated in the absence of the glucocorticoid analog and 24 h prior immunofluorescence staining with anti-CA antibodies the production of MMTV-specific proteins was either induced (A) or not (B) by addition of 10-6 M DEX in the cell culture media. The nuclei of the cells were counterstained with DAPI. (C) Western blot detecting the expression of gp52 and gp36 Env proteins in the second-round infected HS578T cells. Lane 1, non-infected Hs578T cells, NC; lane 2, infected human cells not stimulated with DEX; lane 3, infected human cells in which the expression of MMTV structural proteins was induced by 10-6 M DEX 24 h before protein harvest.
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Figure 4: Detection of expression of MMTV proteins in the infected human cells. (A and B) Infected Hs578T cells were cultured for 5 weeks in the presence of 10-6 M DEX. One week before immunofluorescence staining the cells were cultivated in the absence of the glucocorticoid analog and 24 h prior immunofluorescence staining with anti-CA antibodies the production of MMTV-specific proteins was either induced (A) or not (B) by addition of 10-6 M DEX in the cell culture media. The nuclei of the cells were counterstained with DAPI. (C) Western blot detecting the expression of gp52 and gp36 Env proteins in the second-round infected HS578T cells. Lane 1, non-infected Hs578T cells, NC; lane 2, infected human cells not stimulated with DEX; lane 3, infected human cells in which the expression of MMTV structural proteins was induced by 10-6 M DEX 24 h before protein harvest.

Mentions: To further demonstrate the replication of MMTV in the human cells exposed to MMTV(GR) virus, indirect immunofluorescence imaging of the MMTV core was performed. A marked difference in the numbers of MMTV core protein expressing cells was detected in a time-course experiment. Whereas only a small number of MMTV-positive cells were detected in the third-round infected cells shortly after infection (Figure 2A), by week five all the cells expressed MMTV antigen (Figure 2B, Figure 4A). The increase was strictly DEX dependent. Upon cultivation of the cells in DEX-free medium no increase in the number of CA-positive cells could be observed (Figure 2D).


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

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

Detection of expression of MMTV proteins in the infected human cells. (A and B) Infected Hs578T cells were cultured for 5 weeks in the presence of 10-6 M DEX. One week before immunofluorescence staining the cells were cultivated in the absence of the glucocorticoid analog and 24 h prior immunofluorescence staining with anti-CA antibodies the production of MMTV-specific proteins was either induced (A) or not (B) by addition of 10-6 M DEX in the cell culture media. The nuclei of the cells were counterstained with DAPI. (C) Western blot detecting the expression of gp52 and gp36 Env proteins in the second-round infected HS578T cells. Lane 1, non-infected Hs578T cells, NC; lane 2, infected human cells not stimulated with DEX; lane 3, infected human cells in which the expression of MMTV structural proteins was induced by 10-6 M DEX 24 h before protein harvest.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Detection of expression of MMTV proteins in the infected human cells. (A and B) Infected Hs578T cells were cultured for 5 weeks in the presence of 10-6 M DEX. One week before immunofluorescence staining the cells were cultivated in the absence of the glucocorticoid analog and 24 h prior immunofluorescence staining with anti-CA antibodies the production of MMTV-specific proteins was either induced (A) or not (B) by addition of 10-6 M DEX in the cell culture media. The nuclei of the cells were counterstained with DAPI. (C) Western blot detecting the expression of gp52 and gp36 Env proteins in the second-round infected HS578T cells. Lane 1, non-infected Hs578T cells, NC; lane 2, infected human cells not stimulated with DEX; lane 3, infected human cells in which the expression of MMTV structural proteins was induced by 10-6 M DEX 24 h before protein harvest.
Mentions: To further demonstrate the replication of MMTV in the human cells exposed to MMTV(GR) virus, indirect immunofluorescence imaging of the MMTV core was performed. A marked difference in the numbers of MMTV core protein expressing cells was detected in a time-course experiment. Whereas only a small number of MMTV-positive cells were detected in the third-round infected cells shortly after infection (Figure 2A), by week five all the cells expressed MMTV antigen (Figure 2B, Figure 4A). The increase was strictly DEX dependent. Upon cultivation of the cells in DEX-free medium no increase in the number of CA-positive cells could be observed (Figure 2D).

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