Differential expression of autocrine motility factor receptor (AMFR) mRNA in normal and cancer cells detected by in situ hybridization

The receptor for autocrine motility factor (AMFR) is known to be involved in the process of AMF-mediated cell migration and metastasis. This paper describes the procedures of non-radioactive in situ hybridization (ISH) detection of AMFR mRNA in both paraffin-embedded surgical sections and cultured cells using either biotinylated oligonucleotide probes or digoxigenin-labeled RNA probes. The results showed that the AMFR mRNA was expressed at an enhanced level in hyperplaJstic and malignant tissues of breast and prostate cancer patient surgical specimens, indicating that the elevated AMFR expression was associated with the tissue malignancy Moreover, AMFR mRNA was detected in both normal and earcinoma cells when cultured at a subconfluent density. However, AMFR expression was inhibited in confluent normal (3T3-A31 murine fibroblast and FHs738BL human bladder) cells while it continued to express in carcinoma (J82 human bladder)and metastatic (3T3-M murine fibroblast) cells irrespective of cell density This suggested a cell-cell contact downregulation of AMFR mRNA expression in normal but not in cancer cells. The ISH data obtained in this study are closely consistent with the AMFR protein expression pattern previously reported, implying that the differential expression of AMFR gene may be regulated and controlled at the transcriptional level.

PARTIAL PURIFICATION AND CHARACTERIZATION OF AN AUTOCRINE T SUPPRESSOR FACTOR FROM MURINE LEUKEMIA

The leukemia-associated autoinhibitor (LAI-615) derived from murine leukemia L7811 has been investigated intensively in our laboratory. In the following experiments, the partial purification of LA I-615 has been carried out in addition to the observation of phenotype variations of L7811 leuke-mic cells. The factor was purified over 1306-fold by sequential fractionation with Sephadex G-150 gel filtration, DEAE-cellulose ion exchange chromato-graphy, and Mono Q-fast protein liquid chromato-graphy. The molecular weight of LAI-615 was 68,000 as estimated by gel filtration. LAI-615 was a protein but not glycosylated, and it was suggested LAI-615 be secreted in an autocrine manner. Im-munocytochemical staining showed that the expression of Lyt2 phenotype of L7811 leukemic cells was often coincident with the secretion of LAI-615. Moreover, the physicochemical characteristics of LAI-615 was similar to that of T suppressor factor. Thus it is concluded that LAI-615 may be one of TsF-like factors.

AMF siRNA treatment of keloid through inhibition signaling pathway of RhoA/ROCK1

A keloid(KD)is a benign dermal fibrotic tumor.Treatment of KDs is challenging and the recurrence rate is high;thus,there is an unmet need to explore new target sites and new treatment methods.As a tumor-associated cytokine,autocrine motility factor(AMF)can effectively stimulate the random and directional movement of cells.We first found that AMF was overexpressed in keloid fibroblasts(KFs)and the proliferation and migration of KFs were promoted by AMF stimulation.After treatment with Y-27632,RhoA kinase inhibitor,the proliferation and migration capacity of KFs declined significantly,and type I collagen protein,active RhoA and ROCK1 also were downregulated.In addition,a KD transplantation model was established under the skin of nude mice,with KD intramural injection AMF siRNA,we found that the weight of the KD was smaller than in the control group(P<0.05),KD tissue sections stained by HE and Masson showed that fibers became loose and the blood vessels were visibly reduced.In conclusion,AMF siRNA is expected to be a novel strategy to treat KD by inhibiting signaling pathway of RhoA/ROCK1.