Two-dimensional gel electrophoresis analyses identify nucleophosmin as an estrogen regulated protein associated with acquired estrogen-independence in human breast cancer cells.
We have used two-dimensional gel electrophoresis to identify proteins associated with estrogen-induced proliferation in MCF-7 breast cancer cells and their progression to estrogen-independent proliferation. We compared the total cellular proteins from MCF-7 cells and an estrogen independent derivative of the MCF-7 cells MCF-7/LCC1 (Brünner et al. Cancer Research 1993, 53, 283-290), each grown with and without estradiol. These comparisons reveal seven estrogen-regulated proteins. Three of these proteins (HI-1: 36 kDa/pI 4.5, HI-10: 40 kDa/pI 5.5 and HI-19: 62 kDa/pI 5.0) exhibit a 'progression-like' pattern, being induced by estradiol in MCF-7 cells and constitutively present/upregulated in the MCF-7/LCC1 growing without estradiol. HI-11 (65 kDa/pI 5.5) is strongly induced by estradiol in MCF-7 cells but constitutively downregulated and unresponsive to estradiol in the MCF-7/LCC1 cells. Two proteins exhibit a suppressor pattern and are downregulated by estradiol in the estrogen-dependent MCF-7 cells (HI-3: 44 kDa/pI 4.4 and HI-4: 56 kDa/ pI 5.2) and present in MCF-7/LCC1 cells growing without estradiol at levels comparable to that seen in estrogen-treated MCF-7 cells. One protein (HI-9: 68 kDa/pI 5.5) exhibits a marked estrogen regulated pI shift, rather than changes in abundance. We purified and sequenced the HI-10 protein, which we identified as the nucleolar protein, nucleophosmin (NPM). One- and two-dimensional Western blot analyses of MCF-7/LCC1 cell lysates confirmed that HI-10 is immunoreactive with an antinucleophosmin antibody. Western blotting also confirmed the estrogenic regulation of NPM seen in the initial two-dimensional gel electrophoresis studies. Thus, NPM is induced by estradiol in the MCF-7 cells and upregulated in the MCF-7/LCC1 cells growing without estrogen, clearly associating its expression with an acquired estrogen-independent phenotype. NPM has several potentially important roles in regulating cell function and signaling. It is a substrate for phosphorylation by p34cdc2 kinase, protein kinase C and nuclear kinase II, and a repressor of the transcriptional regulating activities of both the IRF-1 tumor suppressor protein and the YY1 transcription factor. Studies are currently underway to determine which of these NPM functions may be involved in the hormonal progression of breast cancer.
Skaar
T C
TC
Vincent T. Lombardi Cancer Center, Georgetown University Medical School, Washington, DC 20007, USA.
Prasad
S C
SC
Sharareh
S
S
Lippman
M E
ME
Brünner
N
N
Clarke
R
R
D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000090243:Nucleophosmin; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D000090243:Nucleophosmin; D004958:Estradiol