Interstitial cystitis antiproliferative factor (APF) as a cell-cycle modulator

Interstitial cystitis (IC) is a chronic bladder disorder presenting with long-term symptoms of round-the-clock urinary frequency and/or pain in the absence of any other reasonable causation [1]. Although there are multiple hypotheses on the primary cause of IC [2–7], it remains a clinical enigma since there are no definitive data to support these theories [2–7]. A recent breakthrough in IC research has been the discovery of antiproliferative factor (APF) and its association with IC [8]. APF, a peptide purified from the urine of IC patients has been shown to decrease incorporation of thymidine by bladder epithelial cells, decrease levels of urinary heparin-binding epidermal growth factor-like growth factor (HB-EGF) and increase levels of epidermal growth factor (EGF) in vitro which reflects altered levels of these growth factors seen in IC patients [9, 10]. Studies to characterize APF at the molecular level are on-going. To date, the activity corresponds to a single HPLC peak which contains a peptide with a molecular weight less than 3,000 Dalton [10]. Additionally, urine APF activity is found at a higher level in IC vs. non-IC patients [11]. APF is a reliable marker of IC and may be an important factor in the pathogenesis of IC [12]. This study was performed to determine the effect of APF on the cell cycle of bladder epithelial cells so as to better understand its antiproliferative activity.

Primary cultures of normal bladder epithelium were treated in replicate with 10, 20 or 50 μl of APF or mock preparation per 3 ml well. DNA histograms from one replicate of APF treated (10 μl) and one mock treated sample are presented in Figure 1. The histograms clearly show that APF treatment increased the proportion of cells with tetraploid DNA values. There is also a distinct population of cells at the octaploid level. Cell cycle analysis from DNA frequency distributions is relatively straightforward when dealing with a cycling diploid population. The presence of what appears to be a significant cycling tetraploid population in the APF treated cells complicates the analysis. While there are sophisticated algorithms for estimating the cell cycle phases for each of two populations they are largely built on the assumption that the two populations are independent. To determine whether APF may contribute to the formation of tetraploid cells by producing a G2 or M phase block of the cell cycle for both diploid and tetraploid cells, the DNA frequency distributions for all samples were analyzed to extract the proportion of cells with diploid, diploid S, tetraploid, tetraploid S, and octaploid DNA values. Cumulative frequency distributions were calculated and then plotted (Figure 2). Cell sub-populations were derived from these cumulative frequency distributions.

Interstitial cystitis is a chronic bladder disease that, although first described during the 19^th century, today still has no certain etiologies, no specific pathology, and no predictably effective treatment or cure [14]. Hypotheses as to the causes of interstitial cystitis include infection, urinary toxins, increased epithelial permeability, mast cell involvement, neurogenic mechanisms, autoimmune or genetic abnormalities, or a combination of these [2–7]. However, no studies to date have effectively proven any of these mechanisms.

A recent breakthrough for interstitial cystitis has been the discovery of antiproliferative factor [8]. In addition to decreased tritiated thymidine incorporation by bladder epithelial cells treated with APF, explanted bladder epithelial cells from IC patients produce APF, have decreased proliferation, and produce significantly less HB-EGF than bladder cells from controls. Addition of HB-EGF to cell media can overcome the effects of APF on bladder epithelium and cell proliferation [10, 11]. Finally, Chai et al showed that urine APF activity decreases while HB-EGF levels normalize in IC patients following two different IC therapies: bladder hydrodistension [14] and 3rd sacral nerve root stimulation [15]. These findings suggest the intriguing possibilities that bladder epithelial abnormalities in IC may be caused by APF, and that replacement of specific factor(s) altered by APF (such as HB-EGF) may result in normalization of bladder epithelial cell growth [16].

However, the mechanism for APF's action is unclear. It is hypothesized that IC symptoms exacerbate following bladder epithelial damage due to an acute sloughing of epithelial cells. The presence of APF in the urine inhibits appropriate epithelial cells regeneration by regulating autocrine HB-EGF and cell adhesion protein production, resulting in thinning or denudation of the bladder epithelium in IC patients [16].

The goal of the study was to help in the elucidation of APF's mechanism of action. Our primary conclusion from these data is that APF has a profound impact on cell cycle distributions. APF treated cells increase the proportion of cells in the G2M phase, which is likely due to a G2/M block.

Another possible effect of APF that might lead to changes in the bladder epithelia is the production of polyploidy. The appearance of polyploidy is not uncommon in tissue culture and even normal tissue [18, 19]. Polyploidy may result from cellular and then nuclear fusion, or conceivably through mitosis in which cellular DNA is replicated but mitosis and/or cytokinesis fail to occur. Cellular fusion is not uncommon and is even part of normal function in some cell types. After cellular fusion, nuclear fusion probably requires synchronous mitosis, fusion of the mitotic spindle producing two tetraploid daughter cells. This could in turn affect epithelial regeneration as is hypothesized to occur in IC patients. Future studies are needed to help address this issue and to determine if this polyploidy has any significance in APF's mechanism or IC development, or whether it is an unrelated in vitro phenomenon.

The primary bladder epithelial cells used in these experiments were treated with APF or Mock preparations in serum free media. Unlike many other cell types, the lack of exogenous growth factors does not produce a cell cycle blockade. The cells produce sufficient autocrine signaling to maintain a population in a proliferative state, and therefore serum starvation is insufficient to produce cell cycle blockade. Study of synchronized cell populations will greatly facilitate investigation of the mechanism of APF mediated cell cycle changes.

There was an unexpected increase in the proportion of cells with tetraploid DNA content at the highest mock dose. This may be due to the acetonitrile and/or TFA used in the high performance liquid chromatography (HPLC) preparation. There may be a low level of APF in the mock preparation, but no decrease in tritiated thymidine has been observed in similarly controlled experiments, nor can any APF be detected by UV absorbance in the HPLC. Both preparations had equivalent amounts of acetonitrile/TFA so as to control for any affect that these two substances might have on the cells.

Antiproliferative factor has been shown to be a specific protein associated with interstitial cystitis, although its role in the pathogenesis of IC remains unknown. This study shows that APF modulates the cell cycle and in fact might lead to G2 blockade or the production of polyploidy. This would in effect prevent regeneration of bladder epithelial cells, therefore possibly causing the epithelial thinning and/or ulceration associated with IC. Greater understanding of APF's mechanism of action could aid in the diagnoses and treatment of interstitial cystitis.