Development of _targeted therapy for ovarian cancer mediated by a plasmid expressing diphtheria toxin under the control of H19 regulatory sequences

Cell culture

The human ovarian carcinoma cell lines (ES-2, SKOV-3, TOV-112D and OVCAR-3) used in this study were obtained from the American type culture collection (ATCC). Cells were maintained in DMEM-F12 (1:1) medium containing 10% fetal calf serum. For OVCAR-3, 0.01 mg/ml of human insulin was added to the culture medium.

Plasmids and constructs

All the luciferase gene reporter constructs were built from the pGL3 basic (Luc-1) vector (Promega, Madison, WI, USA) which lacks both promoter and enhancer sequences. The construct Luc-H19 contains the reporter gene under the control of the human H19 promoter region from nucleotide -818 to +14 was prepared as described [25]

The Luc-H19 plasmid was digested with Xba I and Nco I and the insert of the luciferase gene (luc) was replaced by the diphtheria toxin A-chain (DT-A) coding region to yield the DTA-H19 construct. Large-scale preparations of the plasmids were performed using the EndoFree Plasmid Mega kit (Quiagen, Germany). All plasmids were modified by replacing the Amp res gene by the Kan res gene.

In vitro transfection and luciferase assay

A total of 1*10^5 cells were plated in a twelve-well Nunc multidish (30 mm). Transient transfections were carried out using the JetPEI cationic polymer transfection reagent (mean molecular weight of 22 kDa; Polyplus, Illkirsh, France). The transfection was carried out according to the manufacturer's instructions using 2 μg of DNA and 3 μl of JetPEI solution to obtain a N/P ratio of 5. Transfection experiments were stopped after 48 h and reporter gene activity was assessed. Luciferase activity was measured using the Promega kit 'Luciferase Assay System' (E-1500; Promega, Madison, USA). Light output was detected using a Lumac Biocounter apparatus. Protein content was measured by the Bio-Rad (Hercules, CA, USA) protein assay reagent, and the results were expressed as light units/μg protein. LucSV40 (Luc-4) was used as a reference for maximal luciferase activity, as it contains the SV40 promoter and enhancer, while Luc-1 lacking regulatory sequences was used as a negative control to determine the basal non-specific luciferase expression, which was found to be negligible. All experiments were carried out in triplicate and the results represent the mean value and standard error was calculated. In all the transfection experiments the measured Luciferase activity is expressed as a percentage of that observed after transfection with the positive control plasmid (Luc-4) alone, to allow normalization of luc activity.

In vitro activity and specificity of the regulatory sequences (cell killing assay)

Cells were cotransfected using 2 μg of the reporter vector Luc-4 and the indicated amounts of the DTA-H19 expression vector using the transfection reagent JetPEI as described above. Cells were also transfected by 2 μg of Luc-4 alone. In vitro activity of the regulatory sequences was determined by calculating the % decrease in the luciferase activity in the cotransfected cells compared with that of the cells transfected only with Luc-4.

RNA isolation and cDNA synthesis

Total RNA was extracted from cell lines or tissues, using the RNA STAT-60™ using total RNA/mRNA isolation reagent (Tel-Test, Inc., Friendswood, TX, USA), according to the manufacturer's instructions. The RNA was treated with RNase-free DNase I (Roche Diagnostics GmbH, Mannheim, Germany) to eliminate any contaminating DNA. The cDNA was synthesized from 2 μg total RNA in 20 μl reaction volume as described [24]

Determination of the level of RNA products of the H19 gene

The PCR reactions were carried out in 25 μl volumes in the presence of 6 ng/μl of each of the forward and the reverse primers using 0.05 units/μl of Taq polymerase (TaKaRa Biomedicals, Japan) according to the manufacturer's instructions. The primer sequences used to amplify the human H19 transcript was (5_-ACTGGAGACTAGGGAGGTCTCTAGCA) upstream and (5_-GCTGTGTGGGTCTGCTCTTTCAAGATG) downstream. The polymerase chain reaction (PCR) was carried out for 30 cycles (98°C for 15 sec, 58°C for 30 sec, and 72°C for 40 sec and finally 72°C for 5 min). The integrity of the cDNA was assayed by RT-PCR analysis using the histone variant, H3.3 or GAPDH as positive control. The products of the PCR reaction were run on 2% agarose in TAE electrophoresis running buffer (40 mM Tris acetate and 2 mM EDTA, pH 8.5), stained by ethidium bromide and visualized by UV.

Human ascites fluid

The Ascitic fluid samples from the peritoneum of patients suffering from ovarian cancer were submitted to this study following approval of the Israeli Ethics Committee. Samples were kindly given to us from the Division of Gynecologic Oncology, Wolfson Medical Center, and from the Department of Gynecology, Hadassah Medical Center. Cells were isolated by using centrifugation of a 15%, 30% and 60% percoll gradient. Cells from the 30% and 60% percoll gradient were used for RNA isolation and ISH analyses. Cells from each isolation (whenever possible), were fixed by 4% PFA on poly-lysine slides and dehydrated, submitted to RNA extraction and seeded in a 750 ml flask.

Immunohistochemistry (IHC)

Following fixation IHC was performed on the isolated ascites cells. The LEVEL1 PEROXIDASE ANTI-PEROXIDASE (PAP) DETECTION SYSTEM and the CA-125 monoclonal antibody (Signet Laboratories Inc. Dedham, MA) were used to detect the CA-125 levels in ascites cells according to manufacture procedure. The fixated cells were rehydrated in PBS*1 at room temperature and were separately incubated with two blocking reagents (hydrogen peroxidase and normal serum) to reduce nonspecific background staining. Cells were then sequentially incubated with three antibody preparations: 1) primary antibody- the CA-125 monoclonal antibody, 2) linking antibody used to bind the primary antibody, 3) labeling antibody, peroxidase labeled mouse immunoglobulin to mark the antigen location. After adding substrate solution cells were counterstained with Mayer's hematoxilin.

Dig-labeled probe synthesis and in situ hybridization

Digoxigenin labeled H19 RNA transcripts were produced by labeling with DIG-11-UTP by SP6, T3, or T7 RNA polymerase in an in vitro transcription reaction (Boehringer, Mannheim, Germany) as described before [26]. The preparation of the sections for in situ hybridization was as described [16]. Finally, the sections were counterstained with 3% Giemsa stain, quickly dried, and mounted in Enthelan. Hybridization was conducted with a sense RNA probe as control to test the specificity of the ISH. The intensity of the hybridization signal was indicated as (+1) for weak, (+2) for moderate and (+3) for strong signals. The distribution of the hybridization signal was referred to as focal (20%–70% of the cells) and defused (>70% of the cells).

Animal heterotopic model for In-vivo DNA based drug

CD-1 or athymic female nude mice (6–8 weeks old, 20–25 g) were used for all the experiments.

All of the surgical procedures and the care given to the animals were approved by the local committee for animal welfare. The histopathological examination of the different tumors was performed in consultation with a trained pathologist.

The level of H19 transcript in ascites from different patients detected by ISH or by RT-PCR

To evaluate the possible use of H19 regulatory sequences for the therapy of ovarian cancer, we determined the level of H19 transcripts in cells from ascites fluid of women patients. Ascitic fluid was collected from the peritoneum of patients carrying ovarian cancer. The ascites cells were separated from contaminating cells (mainly blood cells) on a percoll gradient. The isolated ascites cells were then either fixed on slides for ISH and immunohistochemistry (IHC) analysis (Figure 1A+B), in addition, total RNA was extracted and the level of H19 RNA was determined by RT-PCR analysis (Figure 1C).

We studied the H19 gene expression in 24 different women patients using RT-PCR and ISH techniques. Figure 1D shows the expression of H19 gene in ovarian cancer cells from ascites fluid from different human patients. A semi quantitative scoring system was established to define the levels of H19 expression after ISH (see "Material and Methods").

Figure 1(A+C) shows high levels of H19 transcripts in ascites cells collected from the patients. All patients tested were positive for H19 gene expression. Figure 1A also shows that when H19 transcripts were determined by ISH, a strong positive staining was detected in the cell's cytoplasm. To confirm that the isolated ascites cells originated from ovarian carcinoma, the level of ovarian cancer tumor marker, CA-125, was examined by IHC on some of the slides obtained from the patients (Figure 1B). Positive staining of CA-125 glycoprotein in the ascites cells is shown in Figure 1B which indicates that the cells isolated from the ascites fluid are ovarian cancer cells.

Figure 1D Figure 1D shows that in 23 cases out of 24, ascites cells were positive for H19 transcript (96%). 19 out of 20 (95%) ascites samples examined by RT-PCR showed H19 expression. The ISH analysis showed that in 15 out of 16 (93%) patients the H19 gene was expressed. High and moderate levels of the H19 transcript were detected in 12/15 (74%) samples (samples indicated as 1^I/2^Q were considered as moderate levels of H19). Only 26% (4/15) of the samples tested showed low levels of H19 transcript (indicated as 1^I/1^Q).

Based on these results, we decided to further investigate the use of H19 regulatory sequences for driving toxin gene expression in a therapeutic vector for ovarian cancer.

The level of H19 transcript in human ovarian cancer cell lines

We determined the level of H19 RNA in different human ovarian cancer cell lines. Total RNA was extracted from the cell cultures. The levels of H19 transcripts were detected by RT-PCR analysis in the following cell lines: OVCAR-3, SKOV-3, OV-90, CA-OV3, TOV-112D and ES-2 are shown in Figure 2.

Figure 2 showed different levels of the H19 gene expression. H19 transcripts were detectable in OVCAR-3, SKOV-3, OV-90 and TOV-112D cell lines, while no detectable levels of the H19 transcript were noted in the CA-OV and ES-2 cell lines.

The activity of the human H19 promoter cloned into the Luc-H19 plasmid and the killing effect of the DTA-H19 vector in ascites from patient #1 and in different cell lines

The transcriptional activity of the H19 regulatory sequences cloned into the DTA-H19 plasmid was examined in a variety of cell lines. The luciferase activity induced by the H19 regulatory sequences (Luc -H19 plasmid) was determined in those human cell lines that were previously analyzed for endogenous H19 transcripts expression (Figure 2). Cells were transfected with 2 μg/well of the indicated vectors and luciferase activity was measured by luciferase assay (Figure 3A). Next we also tested the in-vitro killing potential of the DTA-H19 plasmid in the same human ovarian cancer cell lines. OVCAR-3, SKOV-3, TOV-112D and ES-2 cell lines were cotransfected with 2 μg of LucSV40 and the indicated concentrations of DTA-H19 (Figure 3B). Luciferase activity was determined and compared to that of cells transfected with LucSV40 alone. In addition, the killing potential of the DTA-H19 plasmid was tested in ascites from patient #1 (OCC 60%) and in DT-A resistant ovarian carcinoma cell line SKOV-3 as control. Cells were cotransfected with 3 μg of LucSV40 and the indicated concentrations of DTA-H19 (Figure 3C).

The relative reduction of the luciferase activity in the cotransfected cells reflect the level of the H19 driven DT-A expression and thus cell killing.

The results in Figure 3A showed the relative luciferase activity in the different cell lines which measured the H19 regulatory transcriptional activity in each cell line. Extremely high luciferase activity was detected in the TOV-112D cell line while relatively low levels were detected in OVCAR-3, SKOV-3 and ES-2 cell lines. The levels of H19 transcripts in different cell lines (Figure 2) were not always in accordance with the relative luciferase activity as shown in Figure 3A. This can be explained by the existence of additional regulatory sequences found in the endogenous H19 gene which were not cloned into the plasmid containing the human H19 regulatory sequences, or by differential stability of the H19 RNA in different cell lines.

A significant decrease in luciferase activity was detected in the cotransfected cell lines (Figure 3B), and in the cotransfected cells obtained from ascites of a patient with advanced ovarian cancer (Figure 3C). The relative reduction of the luciferase activity in the cotransfected cells is completely dependent on hH19 driven DT-A expression and thus cell killing. The H19 promoter is able to drive the expression of the DT-A gene and thus causing inhibition of protein synthesis and cell death which lead to the reduction of LucSV40 activity. The decrease in each cell line is in a dose-response manner. Reduction in luciferase activity of 30%, 75% and 55% (P < 0.003) was obtained after cotransfection of OVCAR-3, TOV-112D and ES-2 cells respectively, with 2 μg/well of LucSV40 and 0.0125 μg/well of the DT-A expressing plasmid (Figure 3B). On the other hand, the diphtheria toxin resistant cell line, SKOV-3 showed no or very low decrease in luciferase activity (Figure 3B and 3C). Moreover, a positive correlation between luciferase activity induced by H19 regulatory sequences shown in Figure 3A and the reduction in luciferase activity due to DTA expression (Figure 3B) can be noted.

In the cotransfected experiments, as the amount of LucSV40 is much larger than those of DTA-H19 plasmid, one can assume that the decrease of luciferase activity is not due to a competition for cell penetration with the DTA-H19 construct, causing a reduction in the amount of LucSV40 which entered the cells, but is a direct consequence of the H19 promoter driven expression of DT-A.

These results justify the use of a DNA based drug in which a toxin is produced under the control of H19 regulatory sequences.

The level of H19 transcripts in heterotopic subcutaneous tumors

In order to develop a model for heterotopic ovarian tumors, ES-2 ovarian carcinoma cells were subcutaneously injected into the dorsa of 6–7 week old CD-1 female mice. Tumors were developed after 9 days and were dissected 14 days after cell injection. Total RNA was extracted from the frozen tumors. The level of H19 RNA was determined by RT-PCR analysis (Figure 4).

Although no H19 expression was detected in the ES-2 cell line, (Figure 2, line 6), significant H19 RNA was detected in all the developed tumors examined (Figure 4), supporting the role of H19 in tumor growth [12]. Thus, the results shown in Figure 4 indicated that the use of this cell line is suitable to establish an ovarian carcinoma animal model. Moreover, the ES-2 cell line causes rapid development of the tumor.

In-vivo tumor growth inhibition by DTA-H19 vector

We used the DTA-H19 vector for evaluating its therapeutic potential by DT-A expression in-vivo using the animal models for ovarian cancer.

Treatment of heterotopic subcutaneous tumors

The ability of the DTA-H19 to promote cancer cell killing and inhibit tumor growth in-vivo was analyzed. ES-2 cells were subcutaneously injected into the back of 6–7 weeks old athymic female mice in order to develop a model for heterotopic ovarian cancer. 10 days after the subcutaneous cell inoculation, the mice developed measurable heterotopic tumors. Mice were randomly divided into two groups: A DTA-H19 group of 12 mice were intratumoral injected with 25 μg of the DTA-H19 plasmid and another group of 12 mice were intratumoral injected with 25 μg of the control plasmid Luc-H19. Both plasmids were injected as complexed with the transfection reagent jetPEI™ (DTA-H19/PEI and Luc-H19/PEI respectively). The sizes of the tumors were determined before each treatment (Figure 5A), and in-vivo fold increase of the tumor size was calculated (Figure 5B).

Figure 5A shows that while similar tumor volumes in the two groups of mice were measured on day 0 (day of the first treatment), inhibition in the rate of tumor growth was detected after each treatment with DTA-H19/PEI plasmid as compared to the tumor growth of Luc-H19/PEI treated mice (p < 0.034). In addition, Figure 5B shows that 4 injections of DTA-H19/PEI plasmid in two-day intervals were able to inhibit tumor growth by 40% compared to 4 Luc-H19/PEI treatments (P < 0.05).