Response of four human ovarian carcinoma cell lines to all-trans retinoic acid: relationship with induction of differentiation and retinoic acid receptor expression

The response of 4 human ovarian carcinoma cell lines to retinoic acid was found to be related to the histological type and degree of differentiation of these tumor cells. The 2 serous cell lines NIHOVCAR3 and OVCCR1 were the most sensitive to the antiproliferative effect of RA. This inhibition was associated with morphological and biological changes that were indicative of differentiation. The undifferentiated IGROV1 cell line was not affected by RA. Since the effects of RA are thought to be mediated by nuclear retinoic acid receptors (RARs), the expression of RARs in human ovarian cancer cells was studied. RAR alpha was detected as mRNA species of 3.1 and 2.6 kb in all 4 cell lines. RAR beta was not detected in any of the cell lines, while RAR gamma (3 kb) was expressed in all of the ovarian cancer cells but at a very low level in the RA-resistant IGROV1 cells.     

Resistance to apoptosis induced by topoisomerase I inhibitors in multidrug-resistant HL60 leukemic cells

The induction of apoptosis by topoisomerase I inhibitors, camptothecin and SN38, was evaluated in drug-sensitive HL60 and multidrug-resistant (MDR) HL60-Vinc leukemic cells. MDR cells displayed a partial resistance to these apoptotic stimuli and this phenomenon was not modulated by verapamil. Basal free calcium concentrations were similar in both cell sublines and were not modified during treatment. Cytoplasmic pH was more acidic in sensitive cells than in MDR cells. Moreover, a significant acidification was obtained during the early stage of apoptosis in sensitive HL60 cells only. Basal Bcl-2 protein expression was found to be greater in MDR than in sensitive cells and was not modulated by apoptosis inducers. This increase of Bcl-2 in MDR cells could be due to the selection process as vincristine enhances Bcl-2 phosphorylation and expression in HL60 sensitive cells. MDR HL60-Vincristine cells therefore display a resistance to apoptosis induced by non-MDR drugs, possibly by Bcl-2 overexpression and inability of these drugs to mediate intracellular pH changes in these drug-resistant cells.     

Gene expression and neuroblastoma cell differentiation in response to retinoic acid: differential effects of 9-cis and all-trans retinoic acid

Retinoic acid has considerable potential for the chemoprevention and chemotherapy of cancer. Neuroblastoma cells differentiate in response to retinoic acid in vitro, an observation that has led to clinical trials using either the 13-cis or all-trans isomers of retinoic acid. We review the effects of retinoic acid on neuroblastoma, and the potential involvement of nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). 9-cis retinoic acid is a ligand for RXRs, and we review recent data on the differential effects of 9-cis and all-trans retinoic acid on neuroblastoma differentiation and proliferation in vitro, and possible mechanisms of action via hetero- and homodimers of RARs and RXRs. Although there is uncertainty whether or not 9-cis retinoic acid produces its biological effects primarily via RXR homodimers, in vitro data suggest that this isomer of retinoic acid or stable analogues may have considerable potential for the treatment of resistant, disseminated neuroblastoma.     

Differentiation of t(5;17) variant acute promyelocytic leukemic blasts by all-trans retinoic acid

All-trans retinoic acid (ATRA) induces differentiation of acute promyelocytic leukemic (APL) blasts from patients with t(15;17) APL. However, blasts from patients with the t(11;17) variant do not differentiate in response to ATRA. Our group has identified a variant of APL characterized by t(5;17) and expression of the NPM-RAR fusion gene product. From case reports it has been difficult to establish whether ATRA induces clinical responses in patients with this variant. In order to determine whether t(5;17) blasts differentiate with ATRA, we harvested mononuclear bone marrow cells from a patient with t(5;17) APL at time of relapse and cultured them in medium containing ATRA. Morphologic analysis of cytospins after 7 days of culture revealed that 60% of cells in the ATRA-treated culture had differentiated into mature neutrophilic forms, as opposed to less than 1% in the control culture. Seventy-three percent of cells acquired NBT positivity after exposure to ATRA, compared with 1% in the control culture. These results indicate that t(5;17) blasts retain the ability to terminally differentiate in response to retinoic acid.     

Inhibitors of tyrosine phosphorylation induce apoptosis in human leukemic cell lines

Experimental evidence suggests that hematopoietic growth factors promote cell survival by suppressing apoptosis or programmed cell death. Since interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) induce tyrosine phosphorylation of a common set of proteins in the factor-dependent cell line M07e, we have investigated whether growth-factor-induced tyrosine phosphorylation is involved in the promotion of cell survival and suppression of apoptosis. Experiments were carried out with the leukemic cell lines HL-60 and M07e and the tyrosine kinase inhibitors genistein and tyrphostin AG82. Both the tyrosine kinase inhibitors induced apoptosis of HL-60 and M07e cells. This was indicated by the appearance of DNA degradation and morphologic evidence of nuclear condensation and fragmentation. It was also confirmed by flow cytometry of DNA, which showed apoptotic cells as a fraction of cells characterized by a diminished DNA stainability, represented on the DNA frequency histograms as a distinct peak below the G0/G1 population. Kinase inhibitors also reduced the fraction of cells in the S phase of the cell cycle. That tyrphostin specifically inhibited tyrosine kinases was further suggested by the prevention of its effects by the tyrosine phosphatase inhibitor sodium orthovanadate (vanadate), at least during the first 18-24 h of treatment. The incomplete prevention of genistein effects by vanadate suggests that genistein is a less specific inhibitor of tyrosine kinases than tyrphostin, and may also act as an inhibitor of topoisomerase II. Vanadate also prevented apoptosis and reduction of the S phase in M07e cells cultured for 24 h in the absence of growth factors. These results suggest that tyrosine phosphorylation is an essential step in IL-3 and GM-CSF signal transduction. Since in our experimental model the effects of tyrosine kinase inhibition and growth factor deprivation could be reversed by concomitant inhibition of tyrosine phosphatases, it is suggested that a balance between tyrosine kinases and tyrosine phosphatases establishes whether a cell will survive or undergo apoptosis.     

A sequential treatment regimen with melatonin and all-trans retinoic acid induces apoptosis in MCF-7 tumour cells

Neoplastic events are marked by uncontrolled cell proliferation. One major focus of cancer research has been to identify treatments that reduce or inhibit cell growth. Over the years, various compounds, both naturally occurring and chemically synthesized, have been used to inhibit neoplastic cell proliferation. Two such oncostatic agents, melatonin and retinoic acid, have been shown to suppress the growth of hormone-responsive breast cancer. Currently, separate clinical protocols exist for the administration of retinoids and melatonin as adjuvant therapies for cancer. Using the oestrogen receptor (ER)-positive MCF-7 human breast tumour cell line, our laboratory has studied the effects of a sequential treatment regimen of melatonin followed by all-trans retinoic acid (atRA) on breast tumour cell proliferation in vitro. Incubation of hormonally responsive MCF-7 and T47D cells with melatonin (10(-9) M) followed 24 h later by atRA (10(-9) M) resulted in the complete cessation of cell growth as well as a reduction in the number of cells to below the initial plating density. This cytocidal effect is in contrast to the growth-suppressive effects seen with either hormone alone. This regimen of melatonin followed by atRA induced cytocidal effects on MCF-7 cells by activating pathways leading to apoptosis (programmed cell death) as evidenced by decreased ER and Bcl-2 and increased Bax and transforming growth factor beta 1 (TGF-beta1) expression. Apoptosis was reflected morphologically by an increase in the number of lysosomal bodies and perinuclear chromatin condensation, cytoplasmic blebbing and the presence of apoptotic bodies. The apoptotic effect of this sequential treatment with melatonin and atRA appears to be both cell and regimen specific as (a) ER-negative MDA-MB-231 and BT-20 breast tumour cells were unaffected, and (b) the simultaneous administration of melatonin and atRA was not associated with apoptosis in any of the breast cancer cell lines studied. Taken together, the results suggest that use of an appropriate regimen of melatonin and atRA should be considered for preclinical and clinical evaluation against ER-positive human breast cancer.     

Differential retinoic acid radiosensitization of cervical carcinoma cell lines

The potential of retinoic acid as a radiosensitizer was investigated using SiHa and CC-1 human uterine cervical carcinoma cell lines, representative of high- and low-grade lesions, respectively. SiHa was significantly (P < 0.05) radiosensitized, whereas CC-1 was not. Although 48 h of treatment with 5 microM 13-cis-retinoic acid prior to irradiation was sufficient to induce radiosensitization, continuation of treatment after irradiation significantly increased the effect (P < 0.05). Three hypotheses were tested to explain the different responses of the two lines. One hypothesis was that SiHa is more sensitive to retinoic acid than CC-1. Measurement of growth revealed that SiHa was more sensitive to growth inhibition by retinoic acid than CC-1. The second hypothesis was that retinoic acid increases the proportion of G1-phase cells in SiHa but not in CC-1. This was found not to be true, because a retinoic acid treatment schedule that induced radiosensitization did not alter cell cycle distribution profiles in the absence of radiation. The third hypothesis was that retinoic acid alters the cell cycle response of SiHa but not CC-1 to radiation. Postirradiation cell cycle profiles revealed that retinoic acid increased G1 delay in SiHa, whereas CC-1 exhibited no significant G1 delay. Both lines exhibited G2 delays that were unaffected by retinoic acid. In conclusion, radiosensitization of SiHa but not CC-1 may be explained by different sensitivities to retinoic acid and differences in postirradiation cell cycle responses. Radiosensitization at radiation doses used clinically was observed when retinoic acid was administered both before and after irradiation.     

Effects of all-trans retinoic acid and interferon alpha in peripheral neuroectodermal tumor cell cultures and xenografts

Peripheral neuroectodermal tumors (PNET) have an unsatisfactory outcome when treated with standard approaches. Among novel treatments, the use of biological response modifiers has rarely been reported in this group of malignancies. We have previously demonstrated that both all-trans retinoic acid (ATRA) and interferon á (IFNá) can inhibit proliferation of human PNET cells and that ATRA can up-regulate IFNá receptor expression in vitro. In this study we evaluated the anti-tumor effects of ATRA and IFNá in PNET cells in vitro and in a human PNET xenograft model, using CHP100 cells. A synergistic inhibitory effect of ATRA and IFNá was observed on CHP100 cells in vitro. On the contrary, a significant inhibition of tumor growth was observed in mice treated with ATRA alone, whereas neither IFNá nor the combination of ATRA and IFNá, reached a statistically significant anti-tumor effect. Histologic examination of tumors revealed the presence of necrosis upon treatment with IFNá, whereas almost no necrosis, but a more differentiated morphology, confirmed by electron microscopy analysis, was associated with the ATRA containing treatments. Taken together these data show an in vitro and in vivo anti-tumor activity of ATRA in human PNET cells, although no synergism of ATRA and IFNá was observed in our xenograft model.     

Carcinoma cell lines resistant for growth inhibition and apoptosis to retinoic acid are responsive to 4-hydroxy-phenyl-retinamide: correlation with tissue transglutaminase

Retinoic acid (RA)-resistant cell lines are highly malignant. To inhibit the growth of the RA-resistant cells we used 4-HPR, a synthetic retinoid, which may act through alternative signal transduction pathways. 4-HPR induced cell growth inhibition and apoptosis in all RA-sensitive as well as -resistant cells, demonstrating a wider spectrum of potency over RA. 4-HPR induced tissue TGase activity. A tight correlation between the induction of tissue TGase, the inhibition of cell growth, and apoptosis was evident in all eight RA-sensitive cell lines. However, basal TGase differed in the different cells, suggesting inducibility rather than basal levels as the relevant parameter. In sharp contrast to the RA-sensitive cells, RA-resistant cells showed sporadic response to 4-HPR for tissue TGase. The wider spectrum of activity of 4-HPR in inhibiting cell growth and inducing apoptosis makes it a good candidate for the treatment of RA-resistant cancer cells.     

Molecular motion in solid all-trans retinoic acid (vitamin A acid) by proton NMR

Case report on a rape-homicide by manual strangulation involving a 59 year old female. The autopsy disclosed a peculiar cross-shaped superficial incised wound of the abdomen, encircled by 12 stab-wounds, furthermore multiple superficial and deep incised wounds of the genitalia. The injury pattern was in good agreement with the psychiatric interpretation of the sexual behaviour as outlet of severe aggressive impulses originating from non-sexual motives.     

Inhibition of cholesteatoma migration in vitro with all-trans retinoic acid

Retinoids have recently become of interest to clinicians because of their ability to inhibit migration and proliferation of premalignant squamous cells while enhancing growth and proliferation of normal cells. An in vitro investigation was undertaken to determine whether retinoic acid exhibits similar inhibitory effects on cholesteatoma cells. Cholesteatoma specimens were obtained intraoperatively from 10 patients undergoing mastoidectomy or revision mastoidectomy for chronic ear disease. Cholesteatoma explant growth and en mass migration were observed daily, and topographic maps were constructed at various time intervals to quantity rate and direction of explant migration in the presence or absence of all-trans retinoic acid. Before all-trans retinoic acid administration, explants migrated very rapidly (1 to 2 mm/day). A maximum threefold inhibition of migratory rate occurred, with explants exposed to 0.1 micromol/L retinoic acid when compared with controls. A sixfold maximum inhibition was observed at higher retinoic acid concentrations (5 micromol/L). On removal of all-trans retinoic acid, twofold and fourfold increases in migratory rates were observed. The direction of explant migration varied significantly for long periods of time and appeared not to be affected by retinoic acid. This investigation suggests that all-trans retinoic acid has an inhibitory effect on cholesteatoma cell migration. Retinoids may have a role in controlling cholesteatoma disease in the future.     

Vitamin K2 and its derivatives induce apoptosis in leukemia cells and enhance the effect of all-trans retinoic acid

Geranylgeraniol, a polyprenylalcohol composing the side chain of vitamin K2 (VK2), was previously reported to be a potent inducer of apoptosis in tumor cell lines (Ohzumi H et al, J Biochem 1995; 117: 11-13). We examined the apoptosis-inducing ability of VK2 (menaquinone 3 (MK3), MK4 and MK5) and its derivatives such as phytonadione (VK1), as well as polyprenylalcohols with side chains of various lengths including farnesol (C15-OH; FO), geranylgeraniol (C20-OH; GGO), and geranylfarnesol (C25-OH; GFO) toward leukemia cells in vitro. MK3, MK4, MK5 and GFO (at 10 microM) showed a potent apoptosis-inducing activity for all freshly isolated leukemia cells tested and for leukemia cell lines such as NB4, an acute promyelocytic leukemia (APL)-derived cell line and MDS92, a cell line derived from a patient with myelodysplastic syndrome, although there were some differences depending on the cells tested. In contrast, VK1 showed no effect on any of the leukemia cells. The combination of MK5 plus all-trans retinoic acid (ATRA) resulted in enhanced induction of apoptosis in both freshly isolated APL cells and NB4 cells as compared to each reagent alone. These data suggest the possibility of using VK2 and its derivatives for the treatment of myelogenous leukemias, including APL.     

Betulinic acid induces apoptosis in human neuroblastoma cell lines

Neuroblastoma has long been recognized to show spontaneous regression during fetal development and in the majority of stage 4s infants < 1 year of age with disseminated disease. Stage 4s disease regresses with no chemotherapy in 50% of the patients. The mechanism by which this occurs is not understood but may be programmed cell death or apoptosis. Betulinic acid (BA) has been reported to induce apoptosis in human melanoma with in vitro and in vivo model systems. Melanoma, like neuroblastoma, is derived from the neural crest cell. We hypothesised that neuroblastoma cells have the machinery for programmed cell death and that apoptosis could be induced by betulinic acid. Nine human neuroblastoma cell lines were treated in vitro with BA at concentrations of 0-20 micrograms/ml for 0-6 days. Profound morphological changes were noted within 3 days. Cells withdrew their axonic-like extensions, became non-adherent and condensed into irregular dense spheroids typical of apoptotic cell death (ED50 = 14-17 micrograms/ml). DNA fragmentation analysis showed ladder formation in the 100-1200 bp region in 3/3 neuroblastoma cell lines treated with BA for 24-72 h. Thus, apparently BA does induce AP in neuroblastoma in vitro. This model will be utilised to investigate the role of apoptosis-related genes in neuroblastoma proliferation and to determine the therapeutic efficacy of BA in neuroblastoma in vivo.     

Retinoic acid receptor-gamma in human epidermis preferentially traps all-trans retinoic acid as its ligand rather than 9-cis retinoic acid

Brain injury in the premature infant is an extremely important problem, in part because of the large absolute number of infants affected yearly. The 2 principal brain lesions that underlie the neurological manifestations subsequently observed in premature infants are periventricular hemorrhagic infarction and periventricular leukomalacia. The emphases of this article are the neuropathological features, pathogenesis, and potential means of prevention of these 2 lesions. Recent work suggests that the ultimate goal, prevention of the lesions, is potentially achievable. Periventricular hemorrhagic infarction may be avoidable by prevention of germinal matrix-intraventricular hemorrhage, and periventricular leukomalacia by detection of impaired cerebrovascular autoregulation, prevention of impaired cerebral blood flow, and interruption of the cascade to oligodendroglial cell death by such agents as free radical scavengers.     

Expression of NADPH oxidase is induced by all-trans retinoic acid but not by phorbol myristate acetate and 1,25 dihydroxyvitamin D3 in the human promyelocytic cell line NB4

Human promyelocytic cells, NB4, differentiate into neutrophils in response to all-trans retinoic acid (ATRA). It has recently been proposed that NB4 cells have bilineage potential because these cells are also able to differentiate into monocyte/macrophages when exposed to a combination of 1,25-dihydroxyvitamin D3 (VD3) and phorbol myristate acetate (PMA). Differentiation of myeloid cells into neutrophils or monocytes is associated with the acquisition of the O2- producing enzyme, NADPH oxidase, which plays a critical role in microbial killing. In this study, the expression of the components of the NADPH oxidase complex during the differentiation of NB4 cells into neutrophils or macrophages has been investigated. Whereas cells exposed to ATRA were able to produce O2- after 2 days of differentiation, they remain unable to generate O2- when exposed to PMA or PMA + VD3. With the exception of p21rac, none of the other oxidase components was expressed in non-differentiated cells. Addition of ATRA induced the progressive expression and accumulation of p22phox, p91phox, p47phox and p67phox. Compared to the other components, p67phox was expressed late and its expression appeared to correlate most closely with the generation of O2- in the differentiation process. In PMA or PMA + VD3-differentiated NB4 cells, expression of the NADPH oxidase components was incomplete. Therefore, ATRA induced the expression of a functional NADPH oxidase complex in neutrophil-like NB4 cells. In contrast, when NB4 cells are exposed to monocytic differentiating agents, they acquire only part of the phenotypic characteristics of monocytes and lack one of the major phagocytic functionalities, the respiratory burst oxidase.     

Immortal cell lines isolated from heart differentiate to an endothelial cell lineage in the presence of retinoic acid

Previously, we isolated a single line of transgenic mice which develop an enlarged heart due to the expression of the immortalizing gene, polyomavirus large T antigen. Immortal cell lines were isolated from adult transgenic but not from nontransgenic hearts. All of the 24 cell lines expressed vimentin and fibronectin but not desmin or myosin heavy chain. We conclude that the cell lines are of non-muscle origin. Six cell lines were chosen for further study. All six cell lines demonstrate profound morphological and biochemical effects when incubated with 10(-4) M to 10(-7) M retinoic acid. The retinoic acid-treated cell lines showed arrested cellular proliferation and aligned to form rows and vesicle-like structures. Cycloheximide inhibited these retinoic acid-induced changes, indicating a need for continued protein synthesis. Retinoic acid-treated, but not untreated, cells lost expression of vimentin and fibronectin, gained the ability to incorporate acetylated low density lipoprotein, and expressed Factor VIII-related antigen. Retinoic acid did not induce expression of desmin or myosin heavy chain. Incubation of the cell lines with transforming growth factor beta 1, dimethyl sulfoxide, or phorbol esters had no biochemical or morphological effect. We conclude that these cell lines differentiate to an endothelial lineage in the presence of retinoic acid.     

Triiodothyronine blocks potentiation of HL60 monocyte differentiation by anti-inflammatory agents and by steroids and induces apoptosis of all-trans retinoic acid "primed" cells

Sensitivity of the human promyeloid cell line HL60 to physiological differentiating agents [e.g. all-trans retinoic acid (all-trans RA) and 1 alpha, 25-dihydroxyvitamin (D3)] is increased by exposure of cells to "anti-inflammatory agents" (e.g. indomethacin) and to steroids [e.g. medroxyprogesterone acetate (MPA)] and post "priming" with a low dose (10(-8) M) of all-trans RA. Co-treatment of serum free-grown HL60 cells (HL60-ITS) with indomethacin and D3 reduces the dose of D3 required for monocyte differentiation from 10(-7) to 6.25 x 10(-9) M. This potentiating effect was observed to be almost absent when experiments where undertaken using serum-grown HL60 cells (HL60-FCS). The agent present in serum that interferes with indomethacin- and MPA-potentiation of the sensitivity of HL60 cells to D3 has been identified as the thyroid hormone 3,5,3'-L-triiodothyronine (T3). "Priming" of HL60-ITS cells with a low dose of all-trans RA reduces the amount of D3 required for the induction of monocyte differentiation to the same degree as co-treatment with either indomethacin or MPA (to 5 x 10(-9) M). However, the combined effect of all-trans RA "priming" and T3 treatment of HL60-ITS cells was induction of apoptosis. Treatment with either agent alone did not result in increased levels of apoptotic cells. These data reveal that T3 has an important influence on the capacity of HL60 cells to undergo differentiation and can promote apoptosis of these cells. Drug combinations, such as a differentiation potentiating agent, for example, indomethacin or MPA, and a differentiation inducer, for example, all-trans RA or D3, may have important therapeutic significance. Serum levels of T3 would be anticipated to influence the outcome.     

Retinoic acid receptors regulate expression of retinoic acid 4-hydroxylase that specifically inactivates all-trans retinoic acid in human keratinocyte HaCaT cells

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), 7beta-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-Ibeta and/or tumor necrosis factor (TNF)-alpha secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7beta-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1beta secretion. TNF-alpha secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the alpha- or beta-hydroxyl radical position plays a key role in apoptosis and IL-1beta secretion.     

Stromelysin 3 is overexpressed in human pancreatic carcinoma and regulated by retinoic acid in pancreatic carcinoma cell lines

OBJECTIVE: To develop a simple prognostic index for anticipating more precisely the early clinical course of primary superficial bladder cancer. PATIENTS AND METHODS: The prognostic value of patient and tumour characteristics was examined in 333 patients with primary Ta or T1 bladder cancer who participated in a multicentre prospective study already completed. Primary tumour multiplicity, a diameter of > 3 cm, stage T1, and grade 2 or 3 were independent predictors of earlier recurrence in a multivariate analysis. A simplified prognostic index consisted of the number of adverse tumour characteristics (ATCs) initially present. RESULTS: After a median follow-up of 35.3 months, the 60 patients free of ATCs (19%) had a recurrence-free probability at 12 and 24 months of 86% and 69%, respectively, and none experienced progression. Recurrence outcomes deteriorated consistently as the number of ATCs increased among the other three groups. In patients with 3-4 ATCs, the 12- and 24-month recurrence-free probability was as low as 30% and 19%, and recurrence and tumour rates were about 2.6 times higher than in patients free of ATCs; 7% of these patients experienced progression within 35 months of follow-up. CONCLUSION: A prognostic index based on the number of ATCs (primary tumour multiplicity, diameter > 3 cm, stage T1, and grade 2 or 3) is a strong indicator of the clinical course of superficial bladder cancer within 3 years of the first endoscopic resection. This proposal is suggested for discussion and for validation in future studies but if confirmed, this simple prognostic index may greatly help to identify indicators for adjuvant intravesical therapy and to determine the optimal periodicity of control cystoscopy regimens.