Depletion of SUMO ligase hMMS21 impairs G1 to S transition in MCF-7 breast cancer cells.
BACKGROUND
hMMS21 is a human SUMO ligase required for DNA damage repair and mitotic progression in HeLa cervical cancer cells. Owing to the diversity of cancer, we further investigated the effect of hMMS21-depletion on MCF-7 breast cancer cells.


METHODS
hMMS21-depletion was achieved by RNA interference. Cellular hMMS21 and E2F1 mRNA levels were estimated by RT-PCR and real-time PCR. Cell cycle profile was assessed by flow cytometry. Western blot and co-immunoprecipitation were used to determine the protein levels of various factors involved in G1-S transition and CDK2- or CDK4-associated p21 and p27. Kinase activity of cyclin E/CDK2 was measured in anti-cyclin E immunoprecipitate.


RESULTS
hMMS21-depletion induced slower cell growth and G1-S transition. While it had no effect on cyclin D1 or phospho-Rb (S807/811) levels, hMMS21-depletion provoked lower E2F1 levels and cyclin E/CDK2 activity. The decreased cyclin E/CDK2 activity correlated with increased cellular p21(CIP1) levels and CDK2-p21 association. Moreover, ectopic expression of Flag-hMMS21 but not its ligase-inactive mutant rescued the decreased growth rates of hMMS21-depletd cells. Thus, depletion of hMMS21 seems to impair G1-S transition due to lowered E2F1 protein levels and cyclin E/CDK2 activity. The decreased cyclin E/CDK2 activity is probably attributable to its greater association with p21 as a result of increased p21 levels. In addition, hMMS21-mediated sumoylation appears to be involved.


CONCLUSIONS
This study demonstrates that hMMS21 is required for G1-S transition in breast cancer cells and implies that manipulation of hMMS21-mediated sumoylation may alter the growth rates of breast cancer cells.
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China.
Huey-Juin
HJ
Ni
Ying-Nang
YN
Chang
Pu-Hong
PH
Kao
Shin-Pei
SP
Chai
Ya-Hsin
YH
Hsieh
Duo-Hsiang
DH
Wang
Jim C
JC
Fong
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D016193:G1 Phase / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D008025:Ligases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000502:physiology*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
C475820:CCNE1 protein, human; C493956:CDKN1A protein, human; C436291:CDKN1B protein, human; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D015513:Oncogene Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; D050760:Cyclin-Dependent Kinase Inhibitor p27; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D008025:Ligases; C503501:NSMCE2 protein, human