Interleukin-6 is associated with poor prognosis in breast cancer. Expression of GP96, a glucose regulated stress protein, is related to drug resistance in tumor cells. Interleukin 6 has previously been shown to induce GP96 expression in a murine myeloblastic cell line. BT474 or RIDA-MB231 cells were incubated with recombinant Interleukin-B (100 to 750 U/ml) for 24 hr. To establish a time course for GP96 induction, MDA-MB231 cells were incubated with 250 U/ml recombinant interleukin-6 for 0-48 hr. Following incubation, cells were washed twice in phosphate-buffered saline (PBS) and cell lysates were prepared by adding 100 mu l of PBS and freezing at -20 degrees C. GP96 was assessed by immunoblotting. Breast tumor tissue and histologically normal breast tissue were obtained within 1 hr of resection and flash frozen in liquid nitrogen. Tissue was homogenized in ice-cold PBS and cell debris was pelleted by centrifugation at 300g at 4 degrees C for 5 min. Supernatants were collected and assayed for interleukin-6 by ELISA, and GP96 by immunoblotting. Both interleukin-8 (P < 0.001) and GP96 are elevated in breast tumor tissue compared to histologically normal tissue. Interleukin-6 (greater than or equal to 250 U/ml for greater than or equal to 12 hr) induces GP96 in the metastatic breast cancer cell line, MDA-MB231, but has no effect on GP96 levels in the primary cell line, BT474. Elevated interleukin-6 in breast tumors may induce GP96 expression in tumor cells conferring a survival advantage by rendering them resistant to cytotoxic therapy and other forms of stress. (C) 1997 Academic Press.Interleukin-6 is associated with poor prognosis in breast cancer. Expression of GP96, a glucose regulated stress protein, is related to drug resistance in tumor cells. Interleukin 6 has previously been shown to induce GP96 expression in a murine myeloblastic cell line. BT474 or RIDA-MB231 cells were incubated with recombinant Interleukin-B (100 to 750 U/ml) for 24 hr. To establish a time course for GP96 induction, MDA-MB231 cells were incubated with 250 U/ml recombinant interleukin-6 for 0-48 hr. Following incubation, cells were washed twice in phosphate-buffered saline (PBS) and cell lysates were prepared by adding 100 mu l of PBS and freezing at -20 degrees C. GP96 was assessed by immunoblotting. Breast tumor tissue and histologically normal breast tissue were obtained within 1 hr of resection and flash frozen in liquid nitrogen. Tissue was homogenized in ice-cold PBS and cell debris was pelleted by centrifugation at 300g at 4 degrees C for 5 min. Supernatants were collected and assayed for interleukin-6 by ELISA, and GP96 by immunoblotting. Both interleukin-8 (P < 0.001) and GP96 are elevated in breast tumor tissue compared to histologically normal tissue. Interleukin-6 (greater than or equal to 250 U/ml for greater than or equal to 12 hr) induces GP96 in the metastatic breast cancer cell line, MDA-MB231, but has no effect on GP96 levels in the primary cell line, BT474. Elevated interleukin-6 in breast tumors may induce GP96 expression in tumor cells conferring a survival advantage by rendering them resistant to cytotoxic therapy and other forms of stress. (C) 1997 Academic Press.