Increased expression and DNA-binding activity of transcription factor Sp1 in doxorubicin-resistant HL-60 leukemia cells.

Abstract

The processes responsible for the multidrug-resistant (Mdr) phenotype in Adriamycin (doxorubicin)-resistant HL-60 leukemia cells (HL-60/AR) are not defined. Since enhanced transcription of resistance-related proteins is associated with Mdr cells, we sought to determine whether changes in the expression of specific transcription factors were a feature characteristic of the Mdr process. Nuclear extracts were prepared from wild-type and resistant cells and compared for their ability to bind DNA consensus sequences for the transcription factors Spl and NFKB contained in the 5' long terminal repeat region of human immunodeficiency virus type 1. Southwestern (DNA-protein) blots showed a family of DNA-binding proteins of 105 kilodaltons (kDa) that were present only in HL-60/AR cells. Competitive gel shift assays indicated that these factors were related to transcription factor Spl, and immunoblotting with an Spl antibody identified this factor as Spi. DNase footprinting of the promoter region in the human immunodeficiency virus type 1 5' long terminal repeat showed that protection occurred at two Spl sites as well as two NFKB sites and the trans-acting region with nuclear extracts only from resistant cells. Preliminary evidence also suggests that phosphorylation may play a negative regulatory role in the activity of Spl, since calf intestine alkaline phosphatase stimulated the DNAbinding activity of Spl in vitro. These results indicate that HL-60/AR cells contain an abundance of DNAbinding proteins, particularly Spl, which probably interact with other cis-acting regulatory proteins in a cooperative manner.The regulation of eucaryotic transcription occurs through specific cis-acting regulatory elements termed promoters or enhancers, which are composed of distinctive DNA sequence motifs (15,23). Regulatory proteins interact with these elements in a highly specific and sometimes cooperative manner and thus determine which genes will be transcriptionally active. Many of the regulatory proteins are inducible factors (22) that respond to such diverse factors as heavy-metal ions (18, 29), heat shock (26), glucocorticoids (7), phorbol esters (1), and anticancer drugs (20). Thus, the control of positive and negative cis-acting regulatory elements may be a function of inducible DNA sequence-specific proteins in response to an environmental stress.

Constitutive Connective Tissue Growth Factor Expression in Scleroderma Fibroblasts Is Dependent on Sp1

“…Our results are, in fact, consistent with data from other systems showing that Sp1 binding is repressed by phosphorylation and that removal of phosphate groups from Sp1 enhances binding to DNA (33)(34)(35)(36)(37)(38)(39). However, it is possible that phosphorylation of Sp1 or Sp1-like proteins could enhance the ability of Sp1 to form an active transcriptional complex with other nuclear factors.…”

Nuclear Factor-κB Is a Critical Mediator of Ste20-Like Proline-/Alanine-Rich Kinase Regulation in Intestinal Inflammation

“…46,47 The reduced levels of Sp1 binding with TNF-␣ stimulation could also be attributable to TNF-␣-induced Sp1 phosphorylation ( Figure 7B) because Sp1 phosphorylation could reduce Sp1 binding 48 and dephosphorylation of Sp1 has been suggested to enhance Sp1 DNA binding activity. 49 In DSS-treated mice, we found that DSS does not alter overall nuclear levels of Sp1, similar studies showed that TNF-␣ did not change Sp1 expression 30 but increased NF-B (p65) expression. 50 Similarly, DSS treatment can up-regulate the expression of NF-B (p65) in colon epithelial cells, and more importantly, DSS treatment can translocate p65 protein into nucleus as active form, which suggests that the increased expression of SPAK might be attributable to the increased nuclear protein NF-B (p65).…”

Ovine adenovirus (OAV287) lacks a virus-associated RNA gene.

“…Nuclear extracts were prepared from a pre-promyelocytic K562 leukaemia cell line (Borellini et al, 1991) as described previously (Prywes & Roeder, 1986) except that protease inhibitors were added to the hypotonic buffer as well as the nuclear suspension buffer (Borellini et al, 1991) and an additional step of concentrating nuclear extract by ammonium sulphate precipitation (0n33 g\ml) was included (Shapiro et al, 1988). The protein concentration (" 8 mg\ml) was measured using a kit from Bio-Rad (Hercules).…”