Overexpression in colorectal carcinoma of two lysosomal enzymes, CLN2 and CLN1, involved in neuronal ceroid lipofuscinosis.
BACKGROUND
Lysosomal proteases are implicated in cancer progression and metastasis. In the current study, using subtraction cloning for genes that are differentially expressed in metastasis, the authors isolated a clone encoding ceroid lipofuscinosis, neuronal 2 (CLN2), which is a lysosomal serine protease defective in neuronal ceroid lipofuscinosis (NCL). Increased CLN2 activity has been reported in breast carcinoma and the antiapoptotic effect of another causative gene of NCL, ceroid lipofuscinosis, neuronal 1 (CLN1), is known.


METHODS
The mRNA levels of CLN2, CLN1, and cathepsins B, D, H, and L were investigated in colorectal carcinoma patients with different clinical stages using real-time quantitative reverse transcriptase polymerase chain reaction. A polyclonal antibody was raised against a recombinant CLN2 protein for immunoblotting and immunohistochemistry.


RESULTS
The mRNA levels of CLN1 and cathepsins B, D, and L were significantly higher in metastatic lesions than in primary tumors. In the primary tumors, mRNA expressions of CLN2 and cathepsin D were associated with advanced clinical stages (P < .015 and P < .031, respectively). Among the lysosomal enzymes examined, only the mRNA expression of CLN2 in both the primary tumors of all patients and the pT3 tumors was correlated with the presence of liver metastases (P < .0049 and P < .029, respectively). The polyclonal antibody prepared in the current study demonstrated CLN2 overexpression by immunoblotting and immunohistochemistry.


CONCLUSIONS
The results indicate that there is a close correlation between CLN2 and CLN1 expression and colorectal carcinoma progression and metastasis and suggest that they may be potential molecular targets.
Tsukamoto
Toshihiko
T
Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan. tsukamotot@pharm.kitasato-u.ac.jp
Iida
Juri
J
Dobashi
Yoh
Y
Furukawa
Taiji
T
Konishi
Fumio
F
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000626:Aminopeptidases; D002403:Cathepsins; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000235:genetics*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018450:Disease Progression; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D008875:Middle Aged; D009362:Neoplasm Metastasis / Q000503:physiopathology*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D057057:Serine Proteases; D013869:Thiolester Hydrolases; D000091346:Tripeptidyl-Peptidase 1
D008565:Membrane Proteins; D012333:RNA, Messenger; D000091346:Tripeptidyl-Peptidase 1; D013869:Thiolester Hydrolases; C416054:PPT1 protein, human; D002403:Cathepsins; D010450:Endopeptidases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human