Limits...
Identification of androgen-coregulated protein networks from the microsomes of human prostate cancer cells.

Wright ME, Eng J, Sherman J, Hockenbery DM, Nelson PS, Galitski T, Aebersold R - Genome Biol. (2003)

Bottom Line: Using isotope-coded affinity tags and mass spectrometry we identified and quantified the relative abundance levels of 1,064 proteins and found that distinct cellular processes were coregulated by androgen while others were essentially unaffected.Subsequent pharmacological perturbation of the cellular process for energy generation confirmed that androgen starvation had a profound effect on this pathway.Our results provide evidence for the role of androgenic hormones in coordinating the expression of critical components involved in distinct cellular processes and further establish a foundation for the comprehensive reconstruction of androgen-regulated protein networks and pathways in prostate cancer cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Systems Biology, Seattle, WA 98103, USA.

ABSTRACT

Background: Androgens play a critical role in the development of prostate cancer-dysregulation of androgen-regulated growth pathways can led to hormone-refractory prostate cancer. A comprehensive understanding of androgen-regulated cellular processes has not been achieved to date. To this end, we have applied a large-scale proteomic approach to define cellular processes that are responsive to androgen treatment in LNCaP prostate cancer cells.

Results: Using isotope-coded affinity tags and mass spectrometry we identified and quantified the relative abundance levels of 1,064 proteins and found that distinct cellular processes were coregulated by androgen while others were essentially unaffected. Subsequent pharmacological perturbation of the cellular process for energy generation confirmed that androgen starvation had a profound effect on this pathway.

Conclusions: Our results provide evidence for the role of androgenic hormones in coordinating the expression of critical components involved in distinct cellular processes and further establish a foundation for the comprehensive reconstruction of androgen-regulated protein networks and pathways in prostate cancer cells.

Show MeSH

Related in: MedlinePlus

Respiratory chain inhibitors induce higher levels of cell death in AD cells. (a) Light photomicrographs of SS, AD and AS cells after 72 h. (b) Distribution of mitochondria in SS (left), AD (center) and AS (right) cells. (c) Respiratory chain inhibitors rotenone (10 μM), CCCP (50 μM), antimycin A (10 μg/ml) and oligomycin (5 μg/ml) induce higher levels of cell death in AD cells, as assessed by a cell counting assay after a 24 h incubation with the specified drugs. Data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC395736&req=5

Figure 4: Respiratory chain inhibitors induce higher levels of cell death in AD cells. (a) Light photomicrographs of SS, AD and AS cells after 72 h. (b) Distribution of mitochondria in SS (left), AD (center) and AS (right) cells. (c) Respiratory chain inhibitors rotenone (10 μM), CCCP (50 μM), antimycin A (10 μg/ml) and oligomycin (5 μg/ml) induce higher levels of cell death in AD cells, as assessed by a cell counting assay after a 24 h incubation with the specified drugs. Data are representative of three independent experiments.

Mentions: AD cells increased the levels of proteins associated with the 'energy generation' cellular process, suggesting that these cells were metabolically different in the context of mitochondrial function compared with AS cells (Figure 3). We observed that androgen starvation caused LNCaP cells to adopt a distinct neuronal morphology (Figure 4a, middle panel) when compared to steady-state (SS) or AS cells (Figure 4a, left and right panel, respectively), a finding also reported by other investigators [44]. The cellular distribution of mitochondria in AD cells was quite distinct as the mitochondria traversed the length of the long dendritic-like processes (Figure 4b, middle panel), whereas SS and AS cells displayed a more perinuclear mitochondrial localization pattern (Figure 4b, left and right panels, respectively). The coordinated increase in proteins of the 'energy generation' cellular process in AD cells led us to hypothesize that these cells were stressed at the level of the mitochondria. To test whether AD cells had an increased sensitivity to pharmacological agents that specifically disrupt different steps of the Oxphos pathway when compared with AS cells, we incubated SS, AD and AS cells with rotenone (10 μM), carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 μM), antimycin A (10 μg/ml) or oligomycin (5 μg/ml) and determined cell viability 24 hours later (Figure 4c). As shown in Figure 4c, a 24 h exposure to rotenone, which specifically blocks the NADH dehydrogenase of complex I [45] elicited a slightly higher level of cell death in AD (38.1 ± 3.0%) cells when compared to AS (31.8 ± 2.0%) and SS cells (27.2 ± 1.9%). CCCP, a small aliphatic agent that uncouples electron transport and ATP production [46], resulted in higher levels of cell death in AD cells (53.7 ± 1.9%) when compared to AS (41.5 ± .79%) and SS cells (29.0 ± 2.9%). Antimycin, which specifically blocks complex III of the respiratory chain between cytochrome b and cytochrome c1 [47], induced higher levels of cell death in AD cells (50.9 ± 2.4%) when compared with AS cells (31.1 ± 2.6%) or SS cells (19.9 ± 1.1%). Lastly, oligomycin, which blocks ATP synthesis by binding the F0F1-ATPase of complex V of the respiratory chain [48], also induced higher levels of cell death in AD cells (48.5 ± 1.7%) than in AS (24.1 ± 6.6%) and SS cells (15.8 ± 2.3%). These results clearly show that androgen depletion sensitizes LNCaP cells to cell death induced by drugs that perturb the respiratory chain pathway. A number of plausible explanations could explain the increased cell death response of AD cells to mitochondrial poisons. For example, the neuronal phenotype of AD cells, which resembles the neuroendocrine phenotype of some advanced, hormone refractory PCa that arise after long-term androgen ablation therapy [49,50], may utilize different carbon sources that predispose them to activation of the cell death pathway if mitochondrial function is compromised. Although the biochemical mechanism(s) responsible for inducing higher levels of death in AD cells has yet to be defined, we have established a novel relationship between androgen and energy metabolism in LNCaP cells that will require further scientific inquiry. Whether or not this relationship is maintained in other androgen-dependent prostate cancer cell lines is unknown. However, in the context of clinical therapy for prostate carcinoma, applying traditional androgen-ablation methods in combination with drugs that specifically target the mitochondria may increase the therapeutic efficacy of existing treatment approaches.


Identification of androgen-coregulated protein networks from the microsomes of human prostate cancer cells.

Wright ME, Eng J, Sherman J, Hockenbery DM, Nelson PS, Galitski T, Aebersold R - Genome Biol. (2003)

Respiratory chain inhibitors induce higher levels of cell death in AD cells. (a) Light photomicrographs of SS, AD and AS cells after 72 h. (b) Distribution of mitochondria in SS (left), AD (center) and AS (right) cells. (c) Respiratory chain inhibitors rotenone (10 μM), CCCP (50 μM), antimycin A (10 μg/ml) and oligomycin (5 μg/ml) induce higher levels of cell death in AD cells, as assessed by a cell counting assay after a 24 h incubation with the specified drugs. Data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC395736&req=5

Figure 4: Respiratory chain inhibitors induce higher levels of cell death in AD cells. (a) Light photomicrographs of SS, AD and AS cells after 72 h. (b) Distribution of mitochondria in SS (left), AD (center) and AS (right) cells. (c) Respiratory chain inhibitors rotenone (10 μM), CCCP (50 μM), antimycin A (10 μg/ml) and oligomycin (5 μg/ml) induce higher levels of cell death in AD cells, as assessed by a cell counting assay after a 24 h incubation with the specified drugs. Data are representative of three independent experiments.
Mentions: AD cells increased the levels of proteins associated with the 'energy generation' cellular process, suggesting that these cells were metabolically different in the context of mitochondrial function compared with AS cells (Figure 3). We observed that androgen starvation caused LNCaP cells to adopt a distinct neuronal morphology (Figure 4a, middle panel) when compared to steady-state (SS) or AS cells (Figure 4a, left and right panel, respectively), a finding also reported by other investigators [44]. The cellular distribution of mitochondria in AD cells was quite distinct as the mitochondria traversed the length of the long dendritic-like processes (Figure 4b, middle panel), whereas SS and AS cells displayed a more perinuclear mitochondrial localization pattern (Figure 4b, left and right panels, respectively). The coordinated increase in proteins of the 'energy generation' cellular process in AD cells led us to hypothesize that these cells were stressed at the level of the mitochondria. To test whether AD cells had an increased sensitivity to pharmacological agents that specifically disrupt different steps of the Oxphos pathway when compared with AS cells, we incubated SS, AD and AS cells with rotenone (10 μM), carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 μM), antimycin A (10 μg/ml) or oligomycin (5 μg/ml) and determined cell viability 24 hours later (Figure 4c). As shown in Figure 4c, a 24 h exposure to rotenone, which specifically blocks the NADH dehydrogenase of complex I [45] elicited a slightly higher level of cell death in AD (38.1 ± 3.0%) cells when compared to AS (31.8 ± 2.0%) and SS cells (27.2 ± 1.9%). CCCP, a small aliphatic agent that uncouples electron transport and ATP production [46], resulted in higher levels of cell death in AD cells (53.7 ± 1.9%) when compared to AS (41.5 ± .79%) and SS cells (29.0 ± 2.9%). Antimycin, which specifically blocks complex III of the respiratory chain between cytochrome b and cytochrome c1 [47], induced higher levels of cell death in AD cells (50.9 ± 2.4%) when compared with AS cells (31.1 ± 2.6%) or SS cells (19.9 ± 1.1%). Lastly, oligomycin, which blocks ATP synthesis by binding the F0F1-ATPase of complex V of the respiratory chain [48], also induced higher levels of cell death in AD cells (48.5 ± 1.7%) than in AS (24.1 ± 6.6%) and SS cells (15.8 ± 2.3%). These results clearly show that androgen depletion sensitizes LNCaP cells to cell death induced by drugs that perturb the respiratory chain pathway. A number of plausible explanations could explain the increased cell death response of AD cells to mitochondrial poisons. For example, the neuronal phenotype of AD cells, which resembles the neuroendocrine phenotype of some advanced, hormone refractory PCa that arise after long-term androgen ablation therapy [49,50], may utilize different carbon sources that predispose them to activation of the cell death pathway if mitochondrial function is compromised. Although the biochemical mechanism(s) responsible for inducing higher levels of death in AD cells has yet to be defined, we have established a novel relationship between androgen and energy metabolism in LNCaP cells that will require further scientific inquiry. Whether or not this relationship is maintained in other androgen-dependent prostate cancer cell lines is unknown. However, in the context of clinical therapy for prostate carcinoma, applying traditional androgen-ablation methods in combination with drugs that specifically target the mitochondria may increase the therapeutic efficacy of existing treatment approaches.

Bottom Line: Using isotope-coded affinity tags and mass spectrometry we identified and quantified the relative abundance levels of 1,064 proteins and found that distinct cellular processes were coregulated by androgen while others were essentially unaffected.Subsequent pharmacological perturbation of the cellular process for energy generation confirmed that androgen starvation had a profound effect on this pathway.Our results provide evidence for the role of androgenic hormones in coordinating the expression of critical components involved in distinct cellular processes and further establish a foundation for the comprehensive reconstruction of androgen-regulated protein networks and pathways in prostate cancer cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Systems Biology, Seattle, WA 98103, USA.

ABSTRACT

Background: Androgens play a critical role in the development of prostate cancer-dysregulation of androgen-regulated growth pathways can led to hormone-refractory prostate cancer. A comprehensive understanding of androgen-regulated cellular processes has not been achieved to date. To this end, we have applied a large-scale proteomic approach to define cellular processes that are responsive to androgen treatment in LNCaP prostate cancer cells.

Results: Using isotope-coded affinity tags and mass spectrometry we identified and quantified the relative abundance levels of 1,064 proteins and found that distinct cellular processes were coregulated by androgen while others were essentially unaffected. Subsequent pharmacological perturbation of the cellular process for energy generation confirmed that androgen starvation had a profound effect on this pathway.

Conclusions: Our results provide evidence for the role of androgenic hormones in coordinating the expression of critical components involved in distinct cellular processes and further establish a foundation for the comprehensive reconstruction of androgen-regulated protein networks and pathways in prostate cancer cells.

Show MeSH
Related in: MedlinePlus