PAI-1 Expression Is Required for HDACi-Induced Proliferative Arrest in ras-Transformed Renal Epithelial Cells

Malignant transformation of mammalian cells with ras family oncogenes results in dramatic changes in cellular architecture and growth traits. The generation of flat revertants of v-K-ras-transformed renal cells by exposure to the histone deacetylase inhibitor sodium butyrate (NaB) was previously found to be dependent on transcriptional activation of the PAI-1 (SERPINE1) gene (encoding the type-1 inhibitor of urokinase and tissue-type plasminogen activators). NaB-initiated PAI-1 expression preceded induced cell spreading and entry into G1 arrest. To assess the relevance of PAI-1 induction to growth arrest in this cell system more critically, two complementary approaches were used. The addition of a stable, long half-life, recombinant PAI-1 mutant to PAI-1-deficient v-K-ras-/c-Ha-ras-transformants or to PAI-1 functionally null, NaB-resistant, 4HH cells (engineered by antisense knockdown of PAI-1 mRNA transcripts) resulted in marked cytostasis in the absence of NaB. The transfection of ras-transformed cells with the Rc/CMVPAI expression construct, moreover, significantly elevated constitutive PAI-1 synthesis (10- to 20-fold) with a concomitant reduction in proliferative rate. These data suggest that high-level PAI-1 expression suppresses growth of chronic ras-oncogene transformed cells and is likely a major cytostatic effector of NaB exposure.


Introduction
Histone acetyltransferases (HATs) transfer acetyl groups from acetyl CoA to specific lysine residues in the amino terminal histone "tails" to form ε-N-acetyl lysine promoting an "open" or relaxed chromatin structure. Several transcriptional coactivators, including CBP/p300 and SRC, have intrinsic HAT activity [1,2]. Histone deacetylases (HDACs), in contrast, catalyze the removal of acetyl groups on target lysines [3,4] creating a condensed, transcriptionally repressed, chromatin organization [5]. Of the various HDAC inhibitors (HDACi), several exhibit more or less specificity for individual members of the four classes (I-VI) of human HDACs [6,7].

Culture Conditions and Engineered
Cells. The various ras-transformed renal epithelial cell lines used in this study [15,16] grow in serum-free DMEM (at least over the short time frame used in this study; 3-5 days) facilitating assessments of the proliferation-modulating effects of NaB (1-10 mM) and exogenous PAI-1 (0.02-100 nM stable mutant 14-1B, t 1/2 = 145 hours; N150H, K154T, Q319L, M354I) [33] in both the presence and absence of FBS. The derivation of the PAI-1 functionally null knockdown (PAI-1 KD ) 4HH cell line by transfection of a 2.6 kb rat PAI-1 EcoR1/HindIII cDNA fragment (representing nucleotides −118 to +2572) cloned in anti-sense orientation (Rc/CMVIAP) has been described [34,35]. v-rastransformed cells were also transfected with the Rc/CMVPAI sense vector to initiate high-level PAI-1 expression in the absence of NaB or with the empty Rc/CMV construct [32]. Coupled in vitro transcription/translation assay confirmed that a full-length immunoreactive PAI-1 protein was synthesized using the Rc/CMVPAI vector as a template [35]. In some cases, Rc/CMVPAI transfectants were selected with G418 [32]. Cloning strategy and cell line derivation are detailed in the text. c-Ha-ras oncogene-expressing human HaCaT II-4 keratinocytes were described previously [33,36] as were the PAI-1-deficient and reconstituted renal cell lines [35].

Growth Arrest in ras-Transformants Is Restored by Exogenous Exposure to a Long Half-Life PAI-1 Mutant or by Vector-Driven Reconstitution of PAI-1 Expression.
Since targeted suppression of PAI-1 leads to bypass of both replicative senescence and TGF-β-induced growth arrest [23,27], it was important to determine if exogenously-delivered PAI-1 could similarly regulate the proliferative response of PAI-1-deficient ras transformants in the absence of NaB. cells in a concentration-dependent manner with an 80% reduction in final population density after a 5-day exposure to 100 nM PAI-1 ( Figure 5(a)). Indeed, the level of growth inhibition in cultures exposed to 20 nM PAI-1 (45% reduction in population density relative to the corresponding control) (Figure 5(a)) approximated the 47.5% decrease induced by 10 mM NaB even in the presence of serum (Figure 4(b)). Ha-ras-transformed HaCaT cells, which express low levels of PAI-1 in response to EGF [33], were also growth inhibited by exposure to PAI-1 14-1B in the presence of FBS or EGF (Figures 5(b) and 5(c)) largely due to G 1 arrest (Figure 5(c)).
To assess this effect more critically in a genetic context, antisense knockdown (PAI-1 KD ; 4HH) cells (Figure 6(a)), which are resistant to NaB-dependent proliferative inhibition, were incubated in PAI-1-supplemented medium with or without, addition of NaB. Recombinant PAI-1, at a final concentration of 20 nM, effectively suppressed PAI-1 KD cell proliferation; the combination of PAI-1 + NaB did not significantly impact the extent of cytostasis compared to PAI-1 alone (Figure 6(b)). Transient vector-driven re-expression of PAI-1 in Rc/CMVPAI v-ras transfectants ( Figure 6(a)) similarly reduced cell growth relative to cells transfected with the empty Rc/CMV construct ( Figure 6(b)). Mass cultures of Rc/CMVPAI-expressing cells and, in particular, their G418-selected clonal isolates, but not cells transfected with Rc/CMV without the 2.6 kb PAI-1 cDNA insert, had significant numbers of very well-spread cells (a hallmark of the growth arrest phenotype in renal epithelial cells [14][15][16]) compared to Rc/CMV populations. The marked reduction in cell proliferation (Figure 6(b)) and increased spreading in Rc/CMVPAI as compared to Rc/CMV transfectants correlated with an approximately 22-fold increase in PAI-1 expression.
(3TP-Lux reporter)-dependent transcription, and increased the incidence of senescent epithelial cells [49]. The present findings are consistent with these and previous data that TGF-β-initiated growth inhibition as well as senescence arrest is PAI-1-dependent [23,27] and establish, moreover, PAI-1 as a mediator of NaB-initiated cytostasis. Whether this response can be adapted for directed "senescence therapy" of human cancers, remains to be assessed. NaB upregulates the cell cycle inhibitors p21 WAF1/CIP1 and p16 INK4A in human fibroblasts although targeted disruption of p21 only weakly impacted HDACi-induced senescencelike growth arrest. p53 −/− mouse embryo fibroblasts (MEFs), moreover, are resistant to NaB-initiated cytostasis indicating that this tumor suppressor is a major senescence determinant in MEFs [50], and NaB-mediated apoptosis in human melanoma cells is p53-dependent [51]. Indeed, nutlin-3, an MDM2 inhibitor which restores p53 function in tumor cells that retain a wild-type p53, cooperate with several HDACis (including NaB) to induce cell death in p53 wild-type tumor cell lines but not in p53-null PC-3 prostatic carcinoma likely by HDACi-induced p53 hyperacetylation and/or MDM2/MDM4 downregulation [52]. This may be dependent, in part, on the extent of increased p53 expression in response to NaB [53]. Similarly, NaB-stimulated p53 transcriptional activity initiated irreversible G 1 /S cell cycle arrest in c-Ha-ras-transformed rat embryo fibroblasts that were p53 wild-type but not in cells with an inactivated p53 [54]. While the actual contribution of p21 versus INK4A/ARF-encoded genes (e.g., p19) in NaB-induced growth arrest is uncertain [55,56], the role of p53 (at least in MEFs) may be more relevant since p53 is required for PAI-1 expression and growth arrest (see [27,57]; and Overstreet et al., in preparation). p53 status, therefore, may be a major aspect of HDACi-induced cell cycle arrest through its transcriptional control of PAI-1 and, thereby, PAI-1dependent cytostasis.
International Journal of Cell Biology 7