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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

Immunofluorescence imaging of MMTV-infected Hs578T cells. The expression of capsid proteins in the third-round infected Hs578T cells was visualized by immunofluorescence staining using a monospecific anti-CA serum. Only a small number of MMTV-positive cells was detected in the third-round infected cells 3 days after infection (A), whereas by week five all the cells expressed MMTV antigen (B). 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 (D). (C) Non-infected Hs578T cells. 24 h prior immunostaining all the cells (A, B, C, D) were grown in medium supplemented with 10-6 M DEX. (Scale bar, 50 μm).
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Figure 2: Immunofluorescence imaging of MMTV-infected Hs578T cells. The expression of capsid proteins in the third-round infected Hs578T cells was visualized by immunofluorescence staining using a monospecific anti-CA serum. Only a small number of MMTV-positive cells was detected in the third-round infected cells 3 days after infection (A), whereas by week five all the cells expressed MMTV antigen (B). 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 (D). (C) Non-infected Hs578T cells. 24 h prior immunostaining all the cells (A, B, C, D) were grown in medium supplemented with 10-6 M DEX. (Scale bar, 50 μm).

Mentions: To demonstrate the production of MMTV-specific structural proteins and further confirm the PCR results, an indirect immunofluorescence staining with mono-specific anti-MMTV-CA polyclonal serum was performed. Consistent with the DNA analysis, the expression of the core protein was detected in the third-round infected cells (Figure 2A). The antigen-expressing cells were grouped in small clusters. These clusters appear to result from division of a single infected cell during the 72-h incubation period or/and possibly by cell-to-cell spread of the virus. The absence of the fluorescence signal in non-infected cells shows the specificity of the reaction (Figure 2C).


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

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

Immunofluorescence imaging of MMTV-infected Hs578T cells. The expression of capsid proteins in the third-round infected Hs578T cells was visualized by immunofluorescence staining using a monospecific anti-CA serum. Only a small number of MMTV-positive cells was detected in the third-round infected cells 3 days after infection (A), whereas by week five all the cells expressed MMTV antigen (B). 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 (D). (C) Non-infected Hs578T cells. 24 h prior immunostaining all the cells (A, B, C, D) were grown in medium supplemented with 10-6 M DEX. (Scale bar, 50 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Immunofluorescence imaging of MMTV-infected Hs578T cells. The expression of capsid proteins in the third-round infected Hs578T cells was visualized by immunofluorescence staining using a monospecific anti-CA serum. Only a small number of MMTV-positive cells was detected in the third-round infected cells 3 days after infection (A), whereas by week five all the cells expressed MMTV antigen (B). 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 (D). (C) Non-infected Hs578T cells. 24 h prior immunostaining all the cells (A, B, C, D) were grown in medium supplemented with 10-6 M DEX. (Scale bar, 50 μm).
Mentions: To demonstrate the production of MMTV-specific structural proteins and further confirm the PCR results, an indirect immunofluorescence staining with mono-specific anti-MMTV-CA polyclonal serum was performed. Consistent with the DNA analysis, the expression of the core protein was detected in the third-round infected cells (Figure 2A). The antigen-expressing cells were grouped in small clusters. These clusters appear to result from division of a single infected cell during the 72-h incubation period or/and possibly by cell-to-cell spread of the virus. The absence of the fluorescence signal in non-infected cells shows the specificity of the reaction (Figure 2C).

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