Another line of DMSO-differentiated HL-60 cells were exposed to cell-free culture supernatants from GST (open bar)- or GST-Tax (closed bar)-preconditioned HL-60 cells for 16 h, and total RNA was extracted from those cells for real-time RT-PCR analysis. DMSO-differentiated HL-60 cells were treated with GST or GST-Tax for 2 h and washed extensively to remove it, and cell-free culture supernatants were collected after 24 h of incubation. (E) Tax-inducible soluble factors played a role in inducing lactoferrin gene expression. The results are means ± SD from triplicate experiments. (Right) Similarly, DMSO-differentiated HL-60 cells were treated with GST (1.5 nM) (open bar), GST-Tax (1.5 nM) plus normal rabbit serum (closed bar), or GST-Tax (1.5 nM) plus anti-Tax serum (hatched bar). (Left) DMSO-differentiated HL-60 cells were incubated with Jurkat cell-free culture supernatant (open bar) or with MT-2 cell-free culture supernatants plus normal rabbit serum (closed bar) or anti-Tax serum (hatched bar). (D) Neutralization of Tax with anti-Tax serum markedly reduced the ability of MT-2 cell-free culture supernatant or GST-Tax preparations to induce lactoferrin gene expression. DMSO-differentiated HL-60 cells were treated with GST (1.5 nM) (open bar), control nonextracted GST-Tax (1.5 nM) (closed bar), or chloroform-extracted GST-Tax (1.5 nM) (hatched bar). (C) Chloroform extraction abolished the GST-Tax effect on lactoferrin gene expression. Expression of lactoferrin mRNA relative to that of tubulin mRNA was calculated for total RNA extracted from DMSO-differentiated HL-60 cells or MCF-7 cells that had been treated with GST (1.5 nM) (open bars), GST (1.2 nM) plus GST-Tax (0.3 nM) (closed bars), or GST-Tax (1.5 nM) (hatched bars) for 16 h, as in panel A. (B) Recombinant Tax protein upregulated lactoferrin gene expression. The results are means ± standard deviations (SD) from three independent experiments. Lactoferrin mRNA expression relative to that of tubulin mRNA in untreated cells is shown as 1. Total RNA was extracted from untreated cells (open bars) or cells that had been cocultured with MT-2 cells through a semipermeable membrane filter (closed bars) or incubated in 50% MT-2 cell-free supernatants (hatched bars) for 2 days and was subjected to real-time RT-PCR of lactoferrin or tubulin gene mRNA. (A) MT-2 cell coculture or MT-2 cell-free culture supernatants upregulated lactoferrin gene expression in DMSO-differentiated HL-60 cells or MCF-7 cells. HTLV-1 Tax induces lactoferrin gene expression. Our findings, in conjunction with our previous study, implicate that mutual interaction between HTLV-1 and lactoferrin would benefit milk-borne transmission of this virus. These results suggest that HTLV-1 infection may be able to induce expression of lactoferrin in a paracrine manner in the lactic compartment. Experiments with Tax mutants, as well as site-directed mutants of the lactoferrin promoter reporters, indicated that the NF-kappaB transactivation pathway is critical for Tax induction of the lactoferrin gene promoter activity in myeloid-differentiated HL-60 cells, but not in MCF-7 cells. In transient-expression assays, Tax transactivated the lactoferrin gene promoter in HL-60 or MCF-7 cells. ![]() Furthermore, extracellularly administered Tax protein also induced lactoferrin gene expression at physiologically relevant concentrations. MT-2 cell coculture could be replaced with cell-free culture supernatants of MT-2 cells to exert the same effect. Coculture with HTLV-1-infected MT-2 cells increased the levels of lactoferrin mRNA in myeloid-differentiated HL-60 cells, as well as MCF-7 cells, models of two probable sources (neutrophils and mammary epithelium) of lactoferrin in breast milk. We now report that HTLV-1 infection can induce lactoferrin gene expression. We have previously demonstrated that lactoferrin, a major milk protein, enhances HTLV-1 replication, at least in part by upregulating the HTLV-1 long terminal repeat promoter. Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia, is transmitted vertically via breastfeeding.
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