Purification of a new intestinal anti-proliferative factor from normal human small intestine.
Previous studies suggest that intestinal cell proliferation may be controlled by endogenous mitosis inhibitors. We describe here the isolation of a protein named intestinal anti-proliferative factor (IAF) from human small intestine. Successive DEAE anion exchange, isoelectric focusing and gel filtration chromatographies led to a purified anti-proliferative protein fraction used to produce antibodies. Using these antibodies as affinity chromatography ligand, IAF was purified from human small intestine cytosolic fraction. IAF was a potent inhibitor of adenocarcinoma colon cells (HT-29 D4 line) DNA synthesis and proliferation with 50% inhibition observed at picomolar concentrations. Analyzed on SDS/PAGE under reducing conditions, this protein migrates with an apparent molecular mass of 120 kDa and amino acid sequence of two internal peptides displays no homology with another listed protein. Cell cycle studies showed that the growth inhibitory effect was maximal between mid G1 and early S phases. Moreover, flow cytometry studies demonstrated that IAF inhibited the progression of HT-29 D4 cells from G1 to S phase. Northern blot analysis using a dipeptidyl peptidase i.v. probe revealed that the growth arrest mediated by IAF was not linked to differentiation processes. By Western blotting with polyclonal antibodies against IAF, we found that this protein was not detected in differentiated colonic carcinoma. Our results suggest that IAF might regulate intestinal cell proliferation.
Lavagna
C
C
Laboratoire de Gastroentérologie et de Nutrition, Faculté de Médecine de Nice Université de Nice-Sophia Antipolis, France.
Poirée
J C
JC
Fournel
S
S
Rampal
P
P
D000970:Antineoplastic Agents / Q000302:isolation & purification* / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects*; D003110:Colonic Neoplasms / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D018819:Dipeptidyl Peptidase 4 / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D007421:Intestine, Small / Q000737:chemistry*; D009363:Neoplasm Proteins / Q000032:analysis; D011506:Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D011506:Proteins; D012333:RNA, Messenger; D018819:Dipeptidyl Peptidase 4