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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

Infection of human breast cell line Hs578T and feline kidney cells, CrFK, with MMTV(GR) virus. (A) Experimental design. Wpi: weeks post infection. (B) The cells infected with MMTV(GR) were monitored for 20 weeks. Genomic DNA was harvested at week one, six and 20 after infection, respectively, and analyzed by PCR for the presence of MMTV sequences. NC: non-infected cells. M: 1 kb marker. (C) Three infectious cycles were performed in Hs578T cells. The cells infected with MMTV(GR) virus are denoted as first infection cycle. The cell culture supernatant from these Hs578T cells was used in a subsequent infection round. Medium from the second-cycle infected Hs578T cells was used for third infection cycle. M: 1 kb marker. (D) Heat inactivation of the MMTV(GR). Where indicated (heat +) was the virus subjected to the heat treatment (60°C for 10 min). NC: non-infected cells, M: 1 kb marker.
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Figure 1: Infection of human breast cell line Hs578T and feline kidney cells, CrFK, with MMTV(GR) virus. (A) Experimental design. Wpi: weeks post infection. (B) The cells infected with MMTV(GR) were monitored for 20 weeks. Genomic DNA was harvested at week one, six and 20 after infection, respectively, and analyzed by PCR for the presence of MMTV sequences. NC: non-infected cells. M: 1 kb marker. (C) Three infectious cycles were performed in Hs578T cells. The cells infected with MMTV(GR) virus are denoted as first infection cycle. The cell culture supernatant from these Hs578T cells was used in a subsequent infection round. Medium from the second-cycle infected Hs578T cells was used for third infection cycle. M: 1 kb marker. (D) Heat inactivation of the MMTV(GR). Where indicated (heat +) was the virus subjected to the heat treatment (60°C for 10 min). NC: non-infected cells, M: 1 kb marker.

Mentions: Both human (Hs578T) and feline (CrFK), cell lines became infected since the MMTV proviral fragment was readily detectable in the PCR assay (Figure 1B). Importantly, heat inactivation of the virus abrogated the infection as no PCR products were detected in both cell types infected with the heat-treated virus (Figure 1D). The infected cells were cultured for five months and proviral DNA could be detected in both cell types throughout the whole duration of the experiment (Figure 1B), demonstrating that the cells became persistently infected. The primer pair used for PCR assay was specific for MMTV sequences since neither human nor feline endogenous retroviral sequences were amplified in reactions performed with uninfected cells (Figure 1B).


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

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

Infection of human breast cell line Hs578T and feline kidney cells, CrFK, with MMTV(GR) virus. (A) Experimental design. Wpi: weeks post infection. (B) The cells infected with MMTV(GR) were monitored for 20 weeks. Genomic DNA was harvested at week one, six and 20 after infection, respectively, and analyzed by PCR for the presence of MMTV sequences. NC: non-infected cells. M: 1 kb marker. (C) Three infectious cycles were performed in Hs578T cells. The cells infected with MMTV(GR) virus are denoted as first infection cycle. The cell culture supernatant from these Hs578T cells was used in a subsequent infection round. Medium from the second-cycle infected Hs578T cells was used for third infection cycle. M: 1 kb marker. (D) Heat inactivation of the MMTV(GR). Where indicated (heat +) was the virus subjected to the heat treatment (60°C for 10 min). NC: non-infected cells, M: 1 kb marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Infection of human breast cell line Hs578T and feline kidney cells, CrFK, with MMTV(GR) virus. (A) Experimental design. Wpi: weeks post infection. (B) The cells infected with MMTV(GR) were monitored for 20 weeks. Genomic DNA was harvested at week one, six and 20 after infection, respectively, and analyzed by PCR for the presence of MMTV sequences. NC: non-infected cells. M: 1 kb marker. (C) Three infectious cycles were performed in Hs578T cells. The cells infected with MMTV(GR) virus are denoted as first infection cycle. The cell culture supernatant from these Hs578T cells was used in a subsequent infection round. Medium from the second-cycle infected Hs578T cells was used for third infection cycle. M: 1 kb marker. (D) Heat inactivation of the MMTV(GR). Where indicated (heat +) was the virus subjected to the heat treatment (60°C for 10 min). NC: non-infected cells, M: 1 kb marker.
Mentions: Both human (Hs578T) and feline (CrFK), cell lines became infected since the MMTV proviral fragment was readily detectable in the PCR assay (Figure 1B). Importantly, heat inactivation of the virus abrogated the infection as no PCR products were detected in both cell types infected with the heat-treated virus (Figure 1D). The infected cells were cultured for five months and proviral DNA could be detected in both cell types throughout the whole duration of the experiment (Figure 1B), demonstrating that the cells became persistently infected. The primer pair used for PCR assay was specific for MMTV sequences since neither human nor feline endogenous retroviral sequences were amplified in reactions performed with uninfected cells (Figure 1B).

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