Catalytically Active Peptidylglycine α-Amidating Monooxygenase in the Media of Androgen-Independent Prostate Cancer Cell Lines

Abstract

Peptidylglycine α-amidating monooxygenase (PAM) converts inactive terminal-glycine prohormones into their activated α-amidated forms. PAM is thought to play a role in the development of antiandrogen drug resistance in prostate cancer (CaP) through PAMactivated autocrine growth. On the basis of the previous finding that many lung cancer cell lines excrete PAM into their culture media, this study investigates PAM levels in media collected from human CaP cell line cultures. Androgen-independent DU145 and PC-3 prostate cancer cell lines exhibited readily detectable levels of PAM activity in extracts and media, whereas the androgen-dependent LNCaP cell line showed little or no activity. Because of the much larger volume of media versus cell extracts, more than 90% of the total PAM activity was located in the media for both the PC-3 and DU145 cell lines, providing a readily accessible source of CaP PAM. A simple, scalable method to obtain PAM from the culture media of androgen-independent human prostate cancer cell lines is described in this article. This approach provides a much easier means of collecting CaP-derived PAM than previously described cell fractionation procedures and should facilitate the investigations of the role and targeting of PAM in hormone-independent CaP. (Journal of Biomolecular Screening 2008:804-809)

Keywords

androgen independent hormone refractory human prostate cancer cell lines peptidylglycine α-amidating monooxygenase (PAM)peptidylglycine α-hydroxylating monooxygenase (PHM)

References

Prigge ST , Mains RE , Eipper BA , Amzel LM : New insights into copper mono-oxygenases and peptide amidation: structure, mechanism and function. Cell Mol Life Sci 2007;57:1236-1259.

Eipper BA , Milgram SL , Husten EJ , Yun HY , Mains RE : Peptidylglycine α-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains. Protein Sci 1993;2:489-497.

Ouafik L. , Stoffers DA , Campbell TA , Johnson RC , Bloomquist BT , Mains RE , et al: The multifunctional peptidylglycine alpha-amidating monooxygenase gene. Mol Endocrinol 1992;6:1571-1584.

Bolkenius FN , Ganzhorn AJ : Peptidylglycine α-amidating mono-oxygenase: neuropeptide amidation as a target or drug design. Gen Pharmacol 1998 ;31:655-659.

Satani M. , Kenichi T. , Sakamoto H. , Harada S. , Kaida Y. , Noguchi M. : Expression and characterization of human bifunctional peptidylglycine α-amidating monooxygenase. Protein Expr Purif 2003;28:293-302.

Braas KM , Harakall SA , Ouafik L. , Eipper BA , May V. : Expression of peptidylglycine alpha-amidating monooxygenase: an in situ hybridization and immunocytochemical study. Endocrinology 1992;130:2778-2788.

Vos ND , Scott FM , Iwai N. , Treston AM : Expression in human lung cancer cell lines of genes of prohormone processing and the neuroendocrine phenotype. J Cell Biochem Suppl 1996;24:257-268.

Iwai N. , Martinez A. , Miller M. , Vos M. , Mulshine JL , Treston AM : Autocrine growth loops dependent on peptidyl α-amidating enzyme as targets for novel tumor cell growth inhibitors. Lung Cancer 1999;23:209-222.

Rocchi P. , Boudouresque F. , Zamora AJ , Muracciole X. , Lechvallier E. , Martin P. , et al: Expression of adrenomedullin and peptide amidation activity in human prostate cancer and in human prostate cancer cell lines. Cancer Res 2001;61:1196-1206.

10.

Jiminez N. , Jongsma J. , Calvo A. , van der Kwast TH , Treston AM , Cuttitta F. , et al: Peptidylglycine α-amidating monooxygenase- and proadrenomedullinderived peptide-associated neuroendocrine differentiation are induced by androgen deprivation in the neoplastic prostate. Int J Cancer 2001;94:28-34.

11.

Jongsma J. , Oomen MH , Noordzij MA , Van Weerden WM , Martens GJM , van der Kwast TH , et al: Kinetics of neuroendocrine differentiation in an androgen-dependent human prostate xenograft model. Am J Pathol 1999;154:543-551.

12.

Yuan T. , Veeramani S. , Lin F. , Kondrikou D. , Zelivianski S. , Igawa T. , et al: Androgen deprivation induces human prostate epithelial neuroendocrine differentiation of androgen-sensitive LNCaP cells. Endocr Relat Cancer 2006;13:151-167.

13.

Jongsma J. , Oomen MHA , Noordzij MA , Romijn JC , van der Kwast TH , Schroder FH , et al: Androgen-independent growth is induced by neuropeptides in human prostate cancer cell lines. Prostate 2000;42:34-44.

14.

Katopodis A. , May S. : A new facile trinitrophenylated substrate for peptide α-amidation and its use to characterize PAM activity in chromaffin granules. Biochem Biophys Res Comm 1988;151:499-505.

15.

Segel IH : Enzyme Kinetics. New York: John Wiley, 1993.

16.

Bell-Parikh LC , Eipper BA , Mains RE : Response of an integral granule membrane protein to changes in pH. J Biol Chem 2001 ;276:29854-29863.

17.

Zhang JH , Chung TD , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73.