Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

Quantitative measurement of VDR was made using a tritiated calcitriol binding technique, described elsewhere [14].

B.Xenograft and Transgenic Models

In the xenograft model treatment, athymic ‘‘nude’’ mice (4 to 6 weeks old) were injected subcutaneously with human RB cells, as previously described [14–16]. Five days later, they were randomized into treatment and control groups. Animals receiving calcitriol, vitamin D2, and 16,23-D3 received intraperitoneal injections of the vitamin D compound in mineral oil five times a week; control animals received mineral oil alone administered intraperitoneally five times a week. Animals receiving 1a-OH-D2 were given the compound dissolved in 0.1 mL coconut oil by oral gavage, while the control animals received coconut oil alone. Administration was continued for 5 weeks, during which tumor size was measured using calipers and individual mouse weights were recorded three times per week. Toxicity was assessed by survival, animal body weight, and clinical appearance. The mice were then killed and the size, tumor volume, and tumor weight were determined. Serum calcium was measured in blood taken from the subclavian artery just prior to time of death. From representative animals in each group, kidneys were evaluated for calcification. These techniques are described in detail elsewhere [15,16].

The transgenic model treatment was as follows: LHb-Tag mice, a wellcharacterized model of RB [9], were used to determine drug effectiveness in inhibiting tumor growth. Eightto ten-week-old mice were randomized by sex and litter into treatment groups and control groups. The presence of the transgene was confirmed by PCR [9]. Animals receiving calcitriol, vitamin D2, and 16,23-D3 received intraperitoneal injections of the vitamin D compound in mineral oil, while control animals received intraperitoneal injections of mineral oil (vehicle) alone. Treatment was administered five times per week. Animals receiving 1a-OH-D2 were given the compound dissolved in 0.1 mL coconut oil by oral gavage, and control animals received coconut oil alone. The treatment schedule was 5 weeks. Toxicity was assessed and serum calcium determined in the manner described for the xenograft model. At the termination of the treatment period, animals were killed and the eyes were enucleated, fixed in 10% neutral buffered formalin, and subjected to routine histological processing. Tumor area and volume measurements for each study have been previously described [18–20,29]. The histopathological appearance of the tumors (degree of retinal involvement and extension to other ocular structures, severity of necrosis, degree of tumor differentiation, number of mitotic figures, and calcium deposits) was scored by a masked reviewer. The extent and character of the inflammation was also graded [18,19].

Typically, one-way ANOVA was used to assess the effect of dose followed by paired t-tests when a significant effect was found. Also typically, the tumor areas were transformed to the log scale before analysis to obtain uniform variability. Please see specific manuscripts for exact methods [18–20,29].

C.Analysis of Tumor Cell Death

Histological methods of detecting DNA fragmentation were used to identify dying cells in tumors. Terminal transferase dUTP end labeling (TUNEL) was carried out

as described previously [21–23] on paraffin sections of xenograft tumors harvested 5 weeks after treatment. Basically, the 30 ends of nicked DNA were labeled by the addition of biotin-conjugated dUTP using terminal deoxynucleotide transferase (TdT). Labeled DNA was identified by reacting the sections with an avidin peroxidase ABC kit followed by staining with diaminobenzidine. Since TUNEL labels degraded nuclei in both apoptotic and necrotic nuclei, we also stained sections using the ligation method of Didenko and Hornsby [24]. This method specifically labels double-strand DNA strand breaks that have 30 overhanging ends, typical of apoptotic but not necrotic cells. A 200-bp DNA fragment, corresponding to the multiple cloning site of the plasmid vector pBK-CMV, was generated by Taq DNA polymerase and PCR using Texas red-conjugated nucleotides. DNA fragments generated by Taq also contain 30 overhanging ends, which can be ligated to DNA fragments in apoptotic nuclei. Labeled DNA was then layered over tissue sections and ligated to nicked DNA using T4 DNA ligase enzyme. After extensive washing, unligated DNA was removed and labeled nuclei were visualized using a fluorescent microscope. With each method, the tissues were viewed using Nomarski interference optics and were not counterstained.

D.Immunohistochemistry

Paraffin-embedded tissue sections were labeled with antibodies to human p53, human p21, human bax, and the Ki-67 antigen. All antibodies were used with essentially the same protocol described previously [23]. Sections were deparaffinized and subjected to antigen retrieval by incubating them in 100 mM TRIS buffer (pH 9.5) at 908C for 10 min (p21, BAX, and MIB-1) or 30 min (p53), followed by 60 min in the same buffer at room temperature. After incubation with primary antibody, the sections were washed and incubated with appropriate biotinylated secondary antibody and stained with the avidin peroxidase ABC kit. Sections were viewed with Nomarski optics and not counterstained.

III.RESULTS OF VITAMIN D EXPERIMENTS

A.The Presence of Vitamin D Receptors in Y-79 RB Cells

In studies carried out in 1988 [14], Scatchard analysis of the receptor assay showed a concentration of 94 fmol of calcitriol receptors per million Y-79 RB cells, or 56,000 receptors per cell. These receptors have a dissocation constant of 1.18 nmol/L.

B.PCR Amplification of VDR cDNA from Human RB Samples

A total of 23 RB samples taken from the enucleated eyes of different patients were analyzed by PCR for the original presence of VDR mRNA. In every sample, examination of the PCR product by agarose gel electrophoresis revealed a predicted 103-bp band corresponding to a targeted portion of the human VDR coding sequence (representative sample shown in Fig. 2, lane 1). The PCR primer pairs used in both the human and mouse experiments span an intron in order to control for residual genomic DNA. Using the same oligonucleotide primers, an identical 103-bp

Figure 2 PCR amplification of vitamin D receptor (VDR) cDNA obtained from human and mouse tissue samples. Lane 1 contains VDR mRNA from a representative section of fresh human retinoblastoma. Lane 2 contains mRNA from human retinoblastoma cell line Y-79. Lane 3 contains mRNA from cultured HL60 human leukemia cells. Lane 4 contains a representative sample of murine intraocular retinoblastoma. Lane 5 shows a predicted 103 bp band for extraorbital metastasis in the mouse model. Lane 6 contains a representative sample of mouse kidney, which is known to express VDR mRNA.

band was amplified from Y-79 RB (Fig. 2, lane 2) and cultured HL60 human leukemia cells, a cell line known to express VDR [26,27] (Fig. 2, lane 3). DNA sequencing of the PCR product from one human RB sample confirmed that the 103~-bp band encoded the expected VDR region. While all 23 samples were positive in this study, there remains a possibility that a future sample may not be positive. This potential variability is reflected a 95% confidence interval of (0.85,1) for the estimated probability of any RB sample being positive. This interval is calculated assuming binomially distributed outcomes.

C.PCR Amplification of VDR cDNA from a Mouse Model of Retinoblastoma

Retinoblastoma samples from five LHb-Tag mice, along with one metastatic tumor sample and one whole kidney, were processed and analyzed essentially as described for the human RB samples. Mouse renal tissue is known to express VDR [27] and therefore serves as a positive control for these experiments. A predicted 103-bp band was visualized following agarose gel electrophoresis for all samples containing intraocular RB (representative sample shown in Fig. 2, lane 4), extraorbital metastasis (Fig. 2, lane 5), and whole kidney (Fig. 2, lane 6). Analogous to the human samples, the size of the band corresponds to a portion of the murine VDR coding sequence targeted by the murine-specific oligonucleotide primers used during PCR.

D.In Vitro Effects of Calcitriol

Concentrations of 10 9 and 10 6 mol/L of calcitriol were effective at inhibiting the growth of the RB cells in vitro (Fig. 3) [14]. This inhibition was statistically significant at 10 6 mol/L of calcitriol on day 6 and 10 9 mol/L of calcitriol on day 9 (p < 0.05). The 10 12 mol/L of calcitriol did not statistically significantly inhibit cell growth. The inhibition of cell growth was dose-dependent on days 3 and 6.

Figure 3 Relative counts of retinoblastoma cells expressed as percentage of control; 10 9 mol/L of calcitriol caused a 15% decrease in cell growth by day 9.

E.Effectiveness and Toxicity of Ergocalciferol in the Athymic Xenograft Model

Mice were treated with a high dose (7.8 mg/kg) of ergocalciferol and a low dose (2.8 mg/kg) of ergocalciferol. The geometric mean diameter (the cube root of length 6 width 6 height) was measured 33 days after treatment was initiated. The effect of ergocalciferol on tumor size is shown in Figure 4A. The survival data are summarized in Figure 4B. Histopathological examination showed a dose-dependent increase in tumor necrosis and calcification in the treated animals. A more detailed description of the results has been published [17].

F.Effectiveness and Toxicity of Calcitriol Treatment in the Athymic Xenograft Model

In this experiment, the treated group received 500 ng/kg of calcitriol. Tumor size was judged on the basis of geometric mean diameter (Fig. 5A). The results after 30 days of treatment are shown in Figure 5B. Histopathological examination showed no difference in calcification or necrosis between the control tumors and those in the treated animals. Further details regarding these results can be found in the original report [28].

Figure 4 (A) Effect of ergocalciferol on growth of Y-79 retinoblastoma xenografts after 28 days of treatment. The low dose is most effective with p < 0.05 compared to calcitriol. Tumors were measured in terms of geometric mean diameter. (B) Survival of control and ergocalciferol-treated Y-79 retinoblastoma xenograft mice after 33 days of treatment.

Figure 5 (A) Effect of calcitriol on growth of Y-79 retinoblastoma xenografts after 28 days of treatment. The low dose is most effective with p < 0.05 compared to controls. Tumors were measured in terms of geometric mean diameter. (B) Survival of control and calcitriol-treated Y-79 retinoblastoma xenograft mice after 33 days of treatment.

G.Effectiveness and Toxicity of Calcitriol in the LHb-Tag Transgenic Model

In studying the antineoplastic effect of calcitriol in LHb-Tag mice, toxicity studies were done using calcitriol doses of 0.05, 0.1, and 0.2 mg, which were compared to controls. In the dose-response studies, a high dose (0.05 mg) and a low dose (0.025 mg) were administered. Tumor size was measured by the mean largest cross-sectional area. The results after 5 weeks of treatment are shown in Figure 6A. The combined survival results for both the dose-response and toxicity studies following 5 weeks of treatment are summarized in Figure 6B.

Histopathological studies showed no increased calcification in the tumors of treated animals. There was, however, greater cell death in the tumors in the low-dose group (p <0.02). More differentiation was noted in the low-dose group (p <0.003), which was manifest by increased numbers of Homer-Wright rosettes and a smaller proportion of the tumor appearing undifferentiated. Additional information regarding these results is given in the original article [18].

H.Effectiveness and Toxicity of 16,23-D3 in the Athymic Xenograft Model

Preliminary experiments determined the most efficacious dose of 16,23-D3 to be 0.5 mg. The effectiveness and toxicity of this drug was evaluated using a negative control that received mineral oil alone and a positive control that received 0.5 mg of calcitriol. Results of tumor size (tumor volume) are given as of the 32nd day of treatment, because after that day there were insufficient calcitriol-treated mice surviving to permit analysis. The results after 5 weeks of treatment are compared in Figure 7A. Survival for the three groups following 5 weeks of treatment is shown in Figure 7B. On histopathological examination, no qualitative morphological difference was found among the three groups with regard to calcification, necrosis, or differentiation. A more detailed description of these results can be found in the original report [15].

I.Effectiveness and Toxicity of 16,23-D3 in the LHb-Tag Transgenic Model

In an initial study of the effectiveness of 16,23-D3 [19], an extremely low dose of this vitamin D analogue was compared to a control (mineral oil). The results are shown in Figure 8A. All animals survived the 5-week treatment schedule.

Subsequent toxicity studies indicated that considerably larger doses of 16,23- D3 were well tolerated by the transgenic mice [20]. Consequently, a second experiment was performed in which the treatment arms included 0.35-, 0.5-, and 0.75-mg doses. These treatment groups were compared to a control group receiving only mineral oil. Tumor size was measured as the average of the square root of the right and left eye tumor area. The results are presented in Figure 8B. Survival data are shown in Figure 8C. On histological examination of the tumors, no difference in appearance was found between the control and treated animals. Further details of results can be found in the original reports [19,20].

Figure 6 (A) Effect of calcitriol on growth of LHb-Tag transgenic retinoblastomas after 5 weeks of treatment. The difference in tumor size was significant between the high dose and control (p <0.008) and the low dose and control (p <0.014). (B) Survival of control and calcitriol treated LHb-Tag transgenic mice after 5 weeks of treatment.