Dermal fibroblasts promote cancer cell proliferation and exhibit fibronectin overexpression in early mycosis fungoides.

BACKGROUND: Mycosis fungoides (MF) is caused by proliferation of malignant T-cells in the skin and may progress to involve blood, lymph nodes, and viscera. While the skin microenvironment is essential for the initiation and progression of MF in early stages, little is known about the impact of skin stroma on the growth and survival of malignant lymphocytes. OBJECTIVE: We investigated the effect of dermal fibroblasts and their product, fibronectin, on the survival and proliferation of malignant MF cells. METHODS: Fibroblasts and malignant MF CD4 T-cells were isolated from skin of patients with early-stage MF. Fibroblast-lymphocyte co-culture experiments and lymphocyte cultures on fibronectin-coated plates were established utilizing the cells derived from lesional skin, blood, and MF cell lines. The survival and proliferation rates of lymphocytes were assessed via Annexin V and carboxyfluorescein succinimidyl ester assays respectively. Additionally, integrin and fibronectin expressions in MF skin were assessed via immunofluorescence. RESULTS: We found that dermal fibroblasts increased the proliferation rates of MF cells, but not normal skin or blood CD4 T-cells. However, fibroblasts did not rescue MF cells from apoptosis in co-cultures. In MF skin, we found an overexpression of a fibronectin isoform not normally found in healthy skin. MF cells expressed fibronectin-binding integrins and adhered to fibronectin but did not exhibit adhesion-mediated survival via fibronectin-integrin interactions. CONCLUSION: Overall, our results suggest a direct role for fibroblasts, independent of fibronectin-mediated adhesion, in promoting MF cell proliferation. These findings have implications in understanding and targeting the malignant skin stromal microenvironment in cutaneous lymphomas.

Significant bias against the ACA triplet in the tmRNA sequence of Escherichia coli K-12.

The toxin MazF in Escherichia coli cleaves single-stranded RNAs specifically at ACA sequences. MazF overexpression virtually eliminates all cellular mRNAs to completely block protein synthesis. However, protein synthesis can continue on an mRNA that is devoid of ACA triplets. The finding that ribosomal RNAs remain intact in the face of complete translation arrest suggested a purpose for such preservation. We therefore examined the sequences of all transcribed RNAs to determine if there was any statistically significant bias against ACA. While ACA motifs are absent from tmRNA, 4.5S RNA, and seven of the eight 5S rRNAs, statistical analysis revealed that only for tmRNA was the absence nonrandom. The introduction of single-strand ACAs makes tmRNA highly susceptible to MazF cleavage. Furthermore, analysis of tmRNA sequences from 442 bacteria showed that the discrimination against ACA in tmRNAs was seen mostly in enterobacteria. We propose that the unusual bias against ACA in tmRNA may have coevolved with the acquisition of MazF.

Functional impact of HIV coreceptor-binding site mutations.

The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.

DC-SIGN binds to HIV-1 glycoprotein 120 in a distinct but overlapping fashion compared with ICAM-2 and ICAM-3.

DC-SIGN is a C-type lectin that binds to endogenous adhesion molecules ICAM-2 and ICAM-3 as well as the viral envelope glycoprotein human immunodeficiency virus, type 1, glycoprotein (gp) 120. We wished to determine whether DC-SIGN binds differently to its endogenous ligands ICAM-2 and ICAM-3 versus HIV-1 gp120. We found that recombinant soluble DC-SIGN bound to gp120-Fc more than 100- and 50-fold better than ICAM-2-Fc and ICAM-3-Fc, respectively. This relative difference was maintained using DC-SIGN expressed on three different CD4-negative cell lines. Although the cell surface affinity for gp120 varied by up to 4-fold on the cell lines examined, the affinity for gp120 was not a correlate of the ability of the cell line to transfer virus. Monosaccharides with equatorial 4-OH groups competed as well as D-mannose for gp120 binding to DC-SIGN, regardless of how the other hydroxyl groups were positioned. Disaccharide competitors and glycan chip analysis showed that DC-SIGN has a preference for oligosaccharides linked in an alpha-anomeric configuration. Alanine-scanning mutagenesis of DC-SIGN revealed that highly conserved residues that coordinate calcium (Asp-366) and/or are involved in both calcium and specific carbohydrate interactions (Glu-347, Asn-349, Glu-354, and Asp-355) significantly compromised binding to all three ligands. Mutating non-conserved residues (Asn-311, Arg-345, Val-351, Gly-352, Glu-353, Ser-360, Gly-361, and Asn-362) minimally affected binding except for the Asp-367 mutant, which enhanced gp120 binding but diminished ICAM-2 and ICAM-3 binding. Conversely, mutating the moderately conserved residue (Gly-346) abrogated gp120 binding but enhanced ICAM-2 and ICAM-3 binding. Thus, DC-SIGN appears to bind in a distinct but overlapping manner to gp120 when compared with ICAM-2 and ICAM-3.