The retinoblastoma (Rb) family consists of the tumor suppressor pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in 235 specimens of lung cancer show the tightest inverse association between the histological grading in the most aggressive tumor types and pRb2/p130. This led us to study a panel of human lung cancers for mutations in the RB2/p130 gene. Mutations in the Rb-related gene RB2/p130 were detected in 11 of 14(78.5%) primary lung tumors by single-strand conformation polymorphism and sequence analysis. A Moloney leukemia virus-based retroviral system was set up, and a comparable viral concentration of 1 × 107 infectious units/ml was obtained. Retrovirus-mediated delivery of wild-type RB2/p130 to the lung tumor cell line H23 potently inhibited tumorigenesis in vitro and in vivo, as shown by the dramatic growth arrest observed in a colony assay and the suppression of anchorage-independent growth potential and tumor formation in nude mice. The tumors transduced with the RB2/p130 retrovirus diminished in size after a single injection, and a 12-fold reduction in tumor growth after RB2/p130 transduction compared with the Pac-transduced tumors (92% reduction, P = 0.003) and lacZ-transduced tumors (93% reduction, P < 0.001) was found to be statistically significant. These findings provide the missing confirmation that RB2/p130 is a “bona fide” tumor suppressor gene and strengthen the hypothesis that it may be a candidate for cancer gene therapy for lung cancer.

The Rb3gene family includes three members: the Rb tumor suppressor Rb/p105,p107, and RB2/p130. Their protein products are also known as the pocket proteins because of a unique structural and functional domain termed the “pocket,” composed of subdomains A and B separated by a spacer region that is highly conserved among each of the proteins (1, 2, 3, 4, 5).

The structural identities of these proteins underlie similar functional properties. All three family members inhibit cell cycle progression in the G1 phase, and each of their phosphorylation profiles varies in a cell cycle-dependent manner (6, 7, 8, 9, 10). Interestingly, these proteins exhibit unique growth-suppressive properties that are cell type specific, suggesting that although the pocket proteins may complement each other, their functions are not fully redundant (11, 12).

Because restoration of pRb function suppresses the neoplastic properties of pRb-deficient cells (13), we have explored the possibility of restoration of the wild-type RB gene as a clinical treatment for human cancers. Rb family member pRb2/p130 may also hold therapeutic potential. RB2/p130 maps to human chromosome 16q12.2, an area in which deletions have been found in several human neoplasms including breast, ovarian, hepatic, and prostatic cancers (14). HONE-1 cells are a human nasopharyngeal carcinoma cell line that expresses RB2/p130 mRNA at a very low level and shows evidence of alterations within the RB2/p130 locus by Southern blot analysis. Introduction of pRb2/p130 into HONE-1 cells causes a significant reduction in cell proliferation and changes cellular morphology (12). We have demonstrated that the G0-G1 phase cell cycle arrest is mediated by pRb2/p130 in many tumor cell lines, including the human T98G glioblastoma cell line, which is resistant to the suppressive effects of both pRb/p105 and p107 (11, 12, 15).

Recent immunohistochemical studies of the expression patterns of the Rb family members (pRb/p105, p107, and pRb2/p130) in 235 specimens of lung cancer suggest an independent role for RB2/p130 in the development and/or progression of human lung carcinoma (16, 17). As reported, the detection of mutations within the RB2/p130 locus in 12 of 14 primary lung cancers greatly strengthens this hypothesis. Because lung cancer is the most common cause of cancer-related deaths in Western countries (18)and the prognosis of lung cancer is most often dismal, the chance that relatively nontoxic RB2/p130 gene therapy may reduce morbidity and prolong survival is worth investigating.

To establish the foundation for the potential future use of RB2/p130 in gene therapy, we have explored an efficient means of delivering RB2/p130 gene to tumor cell lines with retroviral vectors and have detected high levels of RB2/p130 expression and severe growth suppression in targeted tumors. The identification of mutations within the RB2/p130 gene in primary lung tumors may additionally impact upon therapeutics by playing a role in determining diagnosis and/or prognosis, offering a targeted scope for the potential use of RB2/p130 gene therapy in human lung cancer patients.

Plasmids.

The plasmid pHIT60 [CMV-MLV-gag-pol-SV40ori (gag is a capsid protein,pol is reverse transcriptase and integrase, and ori is origin)], pHIT110 [CMV-Neo-SV40ori (neo is the neomycin resistance gene)] pHIT111 [CMV-lacZ-SV40 promoter-Neo-SV40ori (lacZ is the Escherichia coliβ-galactosidasegene)], and pHIT456 [CMV-MLV-amphotropic env-SV40ori (env is the envelope proteins)] have been described previously (19). The plasmids MSCVneoEB and MSCVPac [derived from the MSCV and LN retroviral vectors (pac is the puromycin resistance gene)] have been described previously (20). The full-length cDNA sequence of RB2/p130 was subcloned into the retroviral vectors MSCVneoEB and MSCVPac in sense and antisense orientations. Briefly, the pRb2/p130 cloned in pCDNA3(BamHI-NotI; Ref. 12) was cut with NotI and religated with an oligonucleotide that changed the NotI site to a BamHI site(5′-GGCCGGGGGATCCCCC-3′). The pRb2/p130 BamHI fragment was gel purified and subcloned into BglII-digested MSCVneoEB or BglII-digested MSCVPac in the sense or in the antisense orientation.

Cell Lines.

The human lung adenocarcinoma cell line H23 has been described previously (21). The cell line A549 (human lung carcinoma)was purchased from the American Type Culture Collection (Manassas, VA). The 293T/17 cell line (human renal carcinoma; Ref. 22) was purchased from the American Type Culture Collection upon authorization of Rockefeller University. A549 and H23 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mml-glutamine. The 293T/17 cell line was maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 2 mml-glutamine.

PCR, SSCP, and Sequence Analysis.

The PCR mixture (50 μl) contained genomic DNA at the final concentration of 4 ng/μl, all four deoxynucleotide triphosphates each at 0.2 mm and deoxyadenosine 5′-[α-[35S]thio]triphosphate, 2 units of high-fidelity Taq polymerase (Boehringer Mannheim,Indianapolis, IN), and primers at a final concentration of 0.4μ m. Thirty-five cycles of denaturation (95°C for 1 min), annealing (55°C for 1 min), and extension (72°C for 1 min)were carried out in a thermal cycler (GeneAmp 2400; Perkin-Elmer,Branchburg, NJ), followed by 7 min at 72°C. Primers (exon 19,5′-AGGTCCTATCACCAAGGGTGT-3′; exon 19 rev, 5′-GCTTAGTTACTTCTTCAAGGC-3′;exon 20, 5′-GAGAAAGTTAATATCCTAGCTG-3′; exon 20, rev 5′-GTGAATGGTCCATATATAAATCA-3′; exon 21, 5′-TGGTTTAGCACACCTCTTCAC-3′;exon 21 rev, 5′-GCTTAGCACAAACCCTGTTTC-3′; exon 22,5′-CTGAGCTATGTGCATTTGCA-3′; and exon 22, rev 5′-AAGGCTGCTGCTAAACAGAT-3′) were used to amplify the following fragments for exons 19–22, respectively: 250, 446, 325, and 232 bp.

For SSCP analysis, MDE gel solution (FMC Corp., Rockland, ME) was used as recommended. The PCR products were gel purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA) and used for automated DNA sequencing with the dideoxy nucleotide terminator reaction chemistry on the Applied Biosystem model 373A DNA sequencer.

Transient Transfection, Transduction, and Determination of Viral Titer.

Transient DNA cotransfections were performed on 293T/17 cells by the calcium phosphate precipitation technique (19, 20, 22, 23). The retroviral supernatant was collected 48 h after transfection and filtered through a 0.45 μm (pore size) filters, and the concentration was measured by transducing H23 and A549 cell lines. Viral titers were determined by counting the FITC-positive cells after PRINS labeling.

Western Blotting.

Western blot analysis were performed as described previously (15), with a polyclonal antibody that specifically recognized pRb2/p130 and failed to cross-react with family members pRb/p105 and p107 (15, 16, 17).

Colony Assay.

Equal numbers of cells (5 × 104cells/plate) were plated the day before transduction. Cells were transduced overnight at 37°C with 1 ml of retroviruses carrying the puromycin resistance gene (Pac) alone or in combination with the RB2/p130 ORF. Supernatant from 293T/17 cells transfected with gag/pol and env genes (1 ml) was used as a negative control (empty virus). Cells were selected with puromycin(Sigma Chemical Co., St. Louis, MO; H23 cells at 2 μg/ml and A549 at 4 μg/ml) for 10 days and then were stained with 2% (w/v) methylene blue in 50% ethanol.

Southern Blot.

A549 and H23 cells were transduced with retroviruses carrying either the puromycin resistance gene alone or in combination with the Rb2/p130 ORF. Cells transduced with retroviruses collected from a cotransfection of 293T/17 cells with only the plasmids carrying gag/pol and env genes were used as negative control (empty virus). Cells were harvested 4 and 8 days after transduction. Genomic DNA was prepared (12) and a 425-nucleotide fragment of the puromycin gene was amplified by PCR using the oligonucleotides PUR5(5′-TCACCGAGCTGCAAGAAC-3′) and PUR3 (5′-GTCCTTCGGGCACCTCGA-3′). The same 425-nucleotide PCR product was amplified with the PUR3 and PUR5 primers and the plasmid MSCVPac as template, which served as a positive control. The amplified DNA was electrophoresed in a 1.2% agarose gel and blotted to a nylon membrane (Hybond-N+; Amersham, Arlington Heights, IL). The 425-nucleotide PCR fragment of the puromycin gene was labeled with deoxycytidine 5′[α-[32P]triphosphate (DuPont NEN Boston,MA) and used as probe. The membrane was washed with 0.2% SDS and 2×SSC and exposed to Kodak X-ray film at −70°C.

PRINS.

Four samples of A549 and H23 cells were prepared for the PRINS reaction. Cells were plated on slides at a concentration of 5 × 105 cells/dish and transduced with 20μl, 50 μl, 100 μl, or 1 ml of retroviruses carrying the puromycin resistance gene alone or in combination with the Rb2/p130 ORF in the sense or the antisense orientation. As a negative control, cells were transduced with supernatant collected from a cotransfection of 293T/17 cells with only the plasmids carrying gag/pol and env genes (empty virus).

Samples were fixed in methanol and glacial acetic acid 3:1 (v/v) for 10 min at room temperature and air dried for 12–24 h. The next day, the samples were dehydrated in a series of ethanol solutions (70, 80, and 100%) each for 5 min and air dried.

For the PRINS reaction, the primers PUR3 and PUR5 were used to amplify a stretch of 425 bp in the puromycin resistance gene present in the plasmids MSCVPac and MSCVPac pRb2/p130 sense and antisense, as well. The reactions were performed as described previously (24). The slides were also incubated with an FITC-conjugated anti-digoxigenin antibody (Boehringer Mannheim, Indianapolis, IN) diluted 1:200 into 2×SSC and 2% BSA and used for detection of digoxigenin-11-dUTP incorporation used in the reaction. The samples were also treated with a solution of 1 μg/ml propidium iodide (Sigma) to stain the unlabeled DNA and then washed in water. Slides were then observed, and stained cells were counted and photographed under a confocal microscope. The same process was applied to OCT tumor embedded (Sakura Finetek USA,Inc., Torrance, CA) frozen sections of H23 cells grown in nude mice.

Soft-Agar Colony Formation Assay.

Soft-agar colony formation assays were performed essentially as described previously (13). H23 cells were plated at a density of 1 × 106 per dish in a 10-cm culture dish the day before transduction. Cells were transduced overnight at 37°C with 1 ml of retroviruses carrying the puromycin resistance gene alone or in combination with the RB2/p130 ORF. Cells were selected in medium containing puromycin (2 μg/ml) for 15 days. Equal number of cells (5 × 103cells) for each transduction were seeded in duplicate in 0.3% agar containing puromycin (2 μg/ml) in 60-mm six-well culture dishes. After 20 days of incubation at 37°C, colonies containing at least 50 cells were counted, and the values for duplicate plates were averaged.

Animal Studies.

Animal care and humane use and treatment of mice were in strict compliance with: (a) institutional guidelines;(b) the Guide for the Care and Use of Laboratory Animals(National Academy of Sciences, 1996); and (c) the Association for Assessment and Accreditation of Laboratory Animal Care International. Tumors were generated by the s.c. injection of H23 cells into nude mice (female NU/NU-nuBR outbred,isolator-maintained mice, 4–5 weeks of age from Charles Rivers,Wilmington, MA).

For the ex vivo studies, H23 tumor cells were transduced in culture with 1 × 107 retroviruses per 10-cm diameter dish carrying only the Pac gene or additionally the Pac gene and RB2/p130 ORF and selected for 15 days. Equal numbers of Pac-transduced and RB2/p130-transduced cells(2.5 × 106 cells) were then injected into each dorsal flank of nude mice (two flanks/mouse with three mice/group) and grown for ∼4 weeks, until the control Pac retrovirus tumors reached a volume of 300–400 mm3. This study was repeated under exactly the same conditions.

For in vivo transduction studies, nude mice were injected along each of their dorsal flanks (two flanks/mouse) with 2.5 × 106 H23 cells/flank. After 15 days when the tumors reached a volume of ∼20 mm3, each tumor was transduced with 5 × 106retroviruses carrying the Pac gene alone or the Pac gene and the E. coliβ-galactosidase (lacZ) gene as control or the Pac gene and RB2/p130 ORF with three animals/group by direct injection of 20 μl of retroviral supernatant directly into each of the tumors. After ∼3 weeks, the animals were sacrificed by CO2 asphyxiation when Pac and lacZ retrovirus-transduced tumors reached a size of 300–350 mm3. This study was repeated under exactly the same conditions. Animal weight was monitored weekly. Tumor growth was followed by measuring the longest axis of the tumor and the axis perpendicular to this with a caliper. Tumor volume was calculated with the formula tumor volume = (length) × (width)2/2. The tumors were then excised and weighed before processing. Tissues that would be used for molecular biological analysis were snap frozen in liquid nitrogen and stored at−80°C. Tissues to be sectioned were placed in OTC (Sakura Finetek USA), frozen in liquid nitrogen, and stored at −80°C or preserved in neutral-buffered formalin at 4°C before embedding in paraffin.

Statistical Analysis.

Analyses involved the comparison of tumor weights and volumes between the various groups during specific times. All analyses of tumor weight and volume were done after first transforming to a log10 scale. The reported average weights are thus geometric means. For weights, we used the t-distribution for confidence intervals. The calculation was done for the logarithm of the total tumor weight/mouse and then transformed back to the original scale and divided by two to express the results as the mean weight/tumor. For the confidence interval for the ratios of weights,the intervals were first calculated for the difference of logarithmic weights and then transformed back. The overall Ps for comparing the size difference between tumors generated in the nude mice and transduced with the Pac, lacZ, or RB2/p130 retroviruses either in vivo or ex vivo were based on a ttest with two samples by assuming two unequal variances. All Ps are two-sided. Volumes were also compared by an ANOVA of logarithmic volumes, with exclusion of values where the tumor was palpable but not measurable. The analysis allowed for differences between mice and for the correlation because of repeated measurements(multiple days) on each tumor.

Immunohistochemistry and β-Galactosidase Assay.

Immumohistochemistry was performed with a rabbit polyclonal immune serum anti-COOH-terminal pRb2/p130 (16, 17) or the anti-β-galactosidase (mouse monoclonal antibody; Promega Corp.,Madison, WI) at a dilution of 1:500 or 1:5000, respectively, and the Vectastain ABC kit (rabbit IgG from Vector Laboratories, Burlingame,CA), essentially as described previously (16, 17, 25, 26, 27). The β-galactosidase assay was performed essentially as described previously (28).

Mutational Screening.

Several immunohistochemical studies have suggested that the lack of RB2/p130 protein expression may play a role in tumor formation and/or progression (16, 17, 25) and that this serves as a negative prognostic indicator (16, 17, 25). Characterization of the genomic structure of the RB2/p130 gene permitted mutational screening (29). Because of the immunohistochemical data and the toll of lung cancer on Western society in terms of morbidity and mortality (18), we began this screening process on primary human lung carcinomas. Fourteen primary specimens of high-grade lung carcinoma (9 adenocarcinomas and 5 squamous cell carcinomas) and their control sample-matched peripheral blood from the same patients were randomly selected for molecular analysis. They were screened for mutation by SSCP analysis within exons 19 and 20 (B domain) and exons 21 and 22 (COOH terminus) of the RB2/p130 gene. This region was analyzed because the majority of the point mutations in RB/p105 are located in the COOH-terminal region (30) and because we have found previously that this region of the RB2/p130 gene to be mutated in different types of tumors.4

Fig. 1 shows the SSCP analysis of exons 20, 21, and 22 of the RB2/p130 gene in primary lung tumors compared with the normal placental DNA and with the normal genomic DNA extracted from peripheral blood of the corresponding patient, indicating that the mutations found in the tumors were somatic. Tumoral and matching normal DNAs were sequenced, confirming the presence of mutations in the tumors and conversely wild-type sequence in the matching peripheral DNAs. Multiple PCR analyses were performed on each of the DNA samples including separate SSCP and sequencing data. A minimum of three separate DNA sequencing trials/sample for each exon examined (exons 19–22) were performed to confirm the mutational data, because we did not have RNA or protein extracts available. Because of the clustering of similar mutations for each tumor samples, exons 19–22 were sequenced and were found to be normal unless so stated in Table 1; therefore, each tumor specimen had SSCP and sequence data from other regions of the gene that were wild type as additional controls. Nucleotide sequencing of the tumoral samples from genomic PCR amplification was performed on both strands, confirming the same mutations. Additionally, because the matching peripheral DNA was found to be wild type by nucleotide sequencing in the same exons examined(exons 19–22), we can conclude that those somatic mutations are not the result of a polymorphism.

Both the direct genomic PCR products and the DNA obtained from the reamplification of the excised bands with altered migration patterns from the SSCP gels were sequenced on both strands to identify the actual mutations. Evaluation of the sequencing chromatograms and the SSCP gels was used to determine whether the mutations were heterozygous or homozygous.

The RB2/p130 gene was found to be mutated in exon 21 and/or exon 22 in 11 of 14 tumors analyzed (78.5%; Table 1). Insertion of an adenosine in exon 22 causing a frameshift in either codon 1100 and/or 1084 was found in 2 of 14 (14.2%) and 3 of 14 (21.4%) samples,respectively. This may disrupt the putative bipartite nuclear localization signal of pRb2/p130 that we identified previously to be necessary for the exclusive nuclear expression of pRb2/p130, which is essential for the G0-G1growth-inhibitory function of pRb2/p130 (31). Along this same line, point mutations in codon 1083, which change a potentially critical lysine residue to arginine or threonine, were identified in 5 of 14 samples (35%) examined. An alteration in the critical second position of the 5′ donor splice site of exon 21 from the highly conserved GT to GG (underlined bases show the change) was a common mutation in 7 of the 11 mutated samples (63%;Table 1). This change may serve to inactivate the function of the second RB2/p130 allele by disrupting the normal splicing pattern of the protein.

The insertion of a thymidine between nucleotides 3367 and 3368 was found in two poorly differentiated adenocarcinomas (28%). Mutation in codon 1049, changing a glutamine in a stop codon, was found in 1 of 10(10%) adenocarcinomas screened. Mutations in codon 1079, changing an asparagine to a phenylalanine, were found in 2 of 10 (20%)adenocarcinomas examined. The homozygous insertion of a cytosine between nucleotides 3326 and 3327 (codon 1086) was found in 2 of 10(20%) adenocarcinomas examined. Finally, mutations in codon 1070,changing an arginine to a glycine, were found in 2 (14%) of 14 samples examined.

We next wanted to determine the effects of expressing pRb2/p130 in vivo in lung tumor cell lines. Because mutations were found in human lung primary tumors, a retroviral system for the delivery of the RB2/p130 gene was established because this may be useful in the future for therapeutic purposes.

Retroviral Production and Titration.

Retrovirus-mediated gene transfer of the putative tumor suppressor gene RB2/p130 in lung cancer cells was carried out with a MLV-based system (19, 22, 32, 33, 34). This system allows for the production of high-titer retroviral stocks by transient transfection of human renal carcinoma 293T/17 cells. This cell line is highly transfectable and expresses the SV40 large T antigen (22). The packaging components gag-pol and env of the MLV are placed on two different plasmids that contain the SV40 origin of replication in their backbone. For this reason, these plasmids will be amplified after being transfected in 293T/17 cells by the SV40 large T antigen. In addition, the MLV packaging components are under the control of the strong human CMV immediate-early promoter (hCMVi.e.;Ref. 19). These two features result in an overexpression system that allows for a rapid generation of high-titer, helper virus-free retroviral stocks, which is a critical requirement for efficient transduction of target cells. The RB2/p130 ORF was placed in MSCV-based transfer vectors (20), which contain genetically modified 5′ LTRs, to achieve both high levels and long-term expression of the transgene.

Retroviral titer was determined by counting the FITC-positive cells obtained after PRINS using primers that amplify a 425-nucleotide fragment of the puromycin resistance gene present in the plasmids MSCVPac and MSCVPac pRB2/p130 in the sense or the antisense orientation that were transduced.

Four sets of samples of human H23 and A549 human lung adenocarcinoma cells were prepared for the PRINS reaction. Cells were transduced with 20 μl, 50 μl, 100 μl, or 1 ml of supernatant containing retroviruses carrying the puromycin resistance gene alone or in combination with the RB2/p130 gene in the sense or the antisense orientation. Supernatant collected from a cotransfection of 293T/17 cells with only the plasmids carrying gag/pol and env (empty retroviral vector) was used as a negative control. FITC-positive cells were counted and photographed under a confocal microscope. Ten random fields/slide were scored, and a comparable viral concentration of 1 × 107 infectious units/ml was found among the retroviruses (data not shown).

Gene Expression after Retroviral Transduction.

To compare the transduction efficacy of the two retroviral vectors, we performed a combination of PCR amplification and Southern blot analysis of a 425-bp fragment of the puromycin resistance gene common to the plasmids MSCVPac and MSCVPac-RB2/p130 sense (Fig. 2 a). The puromycin resistance gene was amplified, because it is not present in mammalian cells and because the signals for DNA integration flank the LTR sequences, thus, ensuring integration of the resistance and cassette genes.

A549 cells were transduced with empty retroviral vector (Fig. 2,a, Lane Neg.) or retroviruses carrying either the puromycin resistance gene alone (Lanes 1 and 3) or in combination with RB2/p130 (Lanes 2 and 4). A549 cells were harvested 4 (Lanes 1 and 2) and 8(Lanes 3 and 4) days after transduction, and genomic DNA was prepared. The same primers used in the PRINS reaction were used to amplify a 425-bp stretch of the puromycin resistance gene. The MSCVPac plasmid was used as a positive control (Fig. 2,a, Lane Pos.). The amplified DNA was electrophoresed in a 1.2% agarose gel and blotted to a nylon membrane. The 42-nucleotide fragment of the puromycin resistance gene, was labeled with[α-32P]dCTP and used as a probe. At 4 and 8 days after transduction, a strong signal for the exogenous puromycin resistance gene was detected in cells transduced with viruses carrying the puromycin gene, suggesting that the transduced genes were integrated into the host genome efficiently (Fig. 2 a). Stable integration into the host cell genome should increase the probability of sustained expression over a prolonged period of the transduced cassette gene (RB2/p130 ORF) than that achieved by ectopic expression.

RB2/p130 Retroviral Gene Delivery.

The aggressive human lung carcinoma H23 and A549 cell lines were transduced to assay the biological effects of RB2/p130 retroviral gene delivery. Fig. 2,b is a representative example of the results obtained with the H23 cell line. The cells were transduced with empty virus (mock) or retroviruses carrying the puromycin resistance gene alone (MSCVPac) or in combination with RB2/p130 (MSCV Pac pRb2/p130)and selected with puromycin for 10 days. The cells transduced with the retroviruses transferring the RB2/p130 ORF were severely growth suppressed in both the H23 (Fig. 2 b) and A549 cell lines(data not shown). Previous flow cytometric analysis(fluorescence-activated cell sorter) data showed that overexpression of RB2/p130 is growth suppressive in different cell lines without any toxic effect, such as an increment of apoptotic or necrotic phenomena (15, 35, 36). The cells were also transduced with serial dilutions of the supernatant retroviruses. The growth-suppressive effects of retroviral delivery of the RB2/p130 gene were dose dependent (data not shown).

We next investigated whether this growth-suppressive effect is contingent upon overexpression of pRb2/p130. 293T/17 cells were transfected with plasmids used for producing the retroviruses. Western blot analysis of these cells demonstrated that the plasmids designed to deliver RB2/p130 resulted in elevated protein levels of pRb2/p130 within the packaging cell line itself (data not shown). However, the key question is whether transduction with retroviruses designed to deliver RB2/p130 results in significant induction of pRb2/p130 protein levels in transduced cells. Fig. 2,c depicts the results of transduction of H23 cells. H23 cells were transduced with empty retroviral vector (Lane 1) or retroviruses carrying the neomycin resistance or puromycin resistance genes alone (Lanes 2 and 4) or in combination with the RB2/p130 gene (Lanes 3 and 5),respectively. As depicted in Lanes 3 and 5,transduction with viruses carrying the RB2/p130 ORF leads to 100-fold increase in pRb2/p130 protein levels. As shown by colony assay and Western blot analysis (Fig. 2, b and c),transduction of this aggressive lung tumor cell line with retroviruses carrying the RB2/p130 ORF leads to overexpression of pRb2/p130,resulting in dramatic suppression of the proliferative potential of this cell line compared with cell lines transduced with viruses carrying only the puromycin resistance gene. Similar results were found with the A549 cell line (data not shown).

Effects of Retroviral Transduction Upon the Tumorigenic Potential of Lung Cancer Cell Lines in Vitro.

The neoplastic properties of the H23 cell line after transduction with retroviruses carrying the puromycin resistance gene alone (MSCVPac) or in combination with RB2/p130 (MSCVPac-pRb2/p130) were assessed by testing their ability to form colonies in soft agar. H23 cells were transduced and selected for 15 days in puromycin and then seeded in duplicate plates containing 0.3% agarose and puromycin. After 3 weeks,colonies containing >50 cells were scored. The colony-forming potential of the H23 cells was dramatically suppressed by the delivery of the RB2/p130 transgene compared with that of pooled clones transduced with MSCVPac alone. The individual colony size of the cells transduced with RB2/p130 was decreased by 5–6-fold, in that the RB2/p130 colonies were 82% smaller than the Pac colonies. Additionally, there was a 10-fold reduction or 90.4% decrease in colony number upon RB2/p130 transgene delivery (Table 2).

Effects of Retroviral Transduction Upon the Tumorigenic Potential of Lung Cancer Cell Lines in Vivo.

Because anchorage-independent cell growth in soft agar is often associated with in vivo tumorigenicity, we next tested the effects of retroviral delivery of RB2/p130 on tumor formation in ex vivo and in vivo experiments. H23 tumor cells were transduced in culture with retroviruses carrying only the puromycin resistance gene or additionally the RB2/p130 ORF and selected for 15 days. Equal numbers of puromycin resistance gene-transduced and Rb2/p130-transduced cells (2.5 × 106 cells) were then injected into each flank of nude mice (three mice/group). Transduction with RB2/p130 greatly suppressed the ability of the cells to form tumors in nude mice compared with those cells transduced with the puromycin resistance gene alone. The average weight of the excised tumors from the puromycin resistance gene and RB2/p130 ex vivo transduced tumors at the conclusion of the first experiment was 0.216 g (95% CI,0.086–0.346 g) and 0.012 g (95% CI, 0.005–0.0172 g), respectively,resulting in an ∼20-fold or 95% reduction in tumor-forming potential that was highly statistically significant (P = 0.002; Table 3). This study was repeated under exactly the same conditions, and transduction with retroviruses carrying RB2/p130 again was found to dramatically suppress the tumor-forming potential of the cells in nude mice that was statistically significantly consistent with the previous study (Table 3).

We next examined the ability of in vivo transduction of RB2/p130 to suppress tumor formation and progression. In the first study, nude mice were injected in each dorsal flank with 2.5 × 106 H23 cells/flank, and tumors were allowed to grow for 15 days. After 15 days, the tumors were transduced with retroviruses carrying the puromycin resistance gene alone or with the bacterial β-galactosidase (lacZ) gene or the RB2/p130 cDNA (three animals/group). At the time of retroviral injection, the tumors were on average 23.77 mm3(95% CI, 20–29 mm3), and there were no statistically significant differences between the tumor volumes in each of the three groups (P = 0.93). Transduction of the tumors with the lacZ gene did not statistically significantly affect the growth rate and tumor-forming potential of the cells compared with those transduced with the puromycin resistance gene alone (P = 0.2; Table 4). The in vivo transduction of RB2/p130 greatly suppressed the tumor-forming potential of the H23 cells compared with that of tumors transduced with lacZ and the puromycin resistance gene (Fig. 3). The average tumor weight of the excised tumors transduced with the puromycin resistance gene alone was 0.245 g (95% CI, 0.134–0.356 g),with lacZ was 0.300 g (95% CI, 0.163–0.437 g), and with RB2/p130 was 0.020 g (95% CI, 0–0.082 g), resulting in >12-fold reduction in tumor growth with RB2/p130 transduction compared with the puromycin resistance gene alone (92% reduction; P < 0.001) and lacZ tumors (93% reduction; P < 0.001) that was highly statistically significant (Table 4). The tumors transduced with the RB2/p130 retrovirus diminished in size after a single injection, and four of the six tumors regressed completely. A statistically significant reduction in the volume of the tumors transduced with the RB2/p130 retrovirus by 4.6-fold or 79% compared with those transduced with the puromycin resistance gene and lacZ retroviruses could be seen 1 week after retroviral injection (P < 0.001).

This study was repeated under exactly the same conditions, and transduction with retroviruses carrying RB2/p130 once again was found to dramatically suppress the tumor-forming potential of the cells in nude mice, an observation that was statistically significantly consistent with the previous study.

In the second in vivo study, transduction of the tumors with the lacZ gene again did not statistically affect the growth rate and tumor-forming potential of the H23 cells, compared with those transduced with the puromycin resistance gene alone(P = 0.2; Table 4). The in vivotransduction of RB2/p130 in the second study also suppressed the tumor-forming potential of the H23 cells compared with transduction of lacZ and the puromycin resistance gene(P < 0.001), respectively (Table 4). The average tumor weight of the excised tumors transduced with the puromycin resistance gene alone was 0.249 g (95% CI, 0.118–0.443 g),with lacZ was 0.309 g (95% CI, 0.197–0.421 g), and with RB2/p130 was 0.042 g (95% CI, 0.012–0.098 g), resulting in a 6-fold reduction in tumor growth with RB2/p130 transduction compared with the tumors transduced with the puromycin resistance gene (83% reduction; P = 0.004) and 7.3-fold reduction compared with those transduced with the lacZ (86% reduction; P < 0.001), which were highly statistically significant (Table 4).

The H23 tumors transduced with the puromycin resistance gene and LacZ formed tumors typical of an undifferentiated lung adenocarcinoma, as seen in Fig. 4,a (and data not shown). More than 75% of the tumor cells were highly positive for pRb2/p130 expression by immunohistochemistry in tumors transduced with the RB2/p130 retrovirus (Fig. 4,b). β-Galactosidase activity and immunohistochemistry used to demonstrate the expression of a functional β-galactosidase protein were performed on sections of the lacZ tumors, and the tumors carrying the puromycin resistance gene as a negative controls, to determine the efficiency of in vivo transduction achieved with the lacZretroviruses (data not shown). Transduction in vivo was also confirmed by the PRINS technique on OTC frozen sections of tumor samples from all three groups and, as negative controls, H23 tumors transduced with empty virus or nontransduced tumors, but using primers that amplify a 425-nucleotide fragment of the puromycin resistance gene present in the Pac, lacZ, and RB2/p130 retroviruses. The tumors transduced in vivo with the Pac, lacZ, and RB2/p130 retroviruses were positive for amplification of the puromycin resistance fragment, but the tumors transduced with empty retroviral vector and nontransduced tumors were negative (Fig. 4, d–f, and data not shown). The suppression of tumor formation and progression in the ex vivo and in vivo transduction studies was dependent upon induction of pRb2/p130 expression in the tumor cells, as shown by Western blot analysis of tumor cell lysates (Fig. 4 c and data not shown).

Retroviruses are among the most efficient vector systems for transducing genes into mammalian cells, and they have been used to deliver therapeutic genes in humans (37, 38, 39). The requirement for host cells to actively divide to allow viral genes to integrate into the host genome (40) may be advantageous for cancer gene therapy. This would limit exogenous gene delivery to rapidly proliferating cancer cells but spare delivery to other nonproliferating cells within the affected organ. The transient three-plasmid expression system for the production of high-titer retroviral vectors (19, 22, 32, 33, 34) has greatly facilitated our study. We obtained a viral concentration of 1 × 107 infectious units/ml 48 h after cotransfection of 293T/17 cells. Another major contribution to our study is derived from the MSCV vectors (20). These transfer vectors contain a modified 5′ LTR, to allow high expression of the transgene after transduction, and are ideally suited for preclinical studies in the murine system. The fact that elevated protein levels of pRb2/p130 were detected in the 293T/17 cell line further supports the selection of the transient system, because high levels of pRb2/p130 may hamper or even prevent the selection of a stable efficient producer cell line.

In essence, we have produced an effective system for gene delivery and stable integration of the RB2/p130 gene in vitroand in vivo that produces high levels of its protein product in transduced cells, resulting in dramatic growth arrest,inhibition of anchorage-independent growth, and suppression of tumor formation and tumor progression in vivo of previously rapidly proliferating aggressive human tumor cell lines. Recent immunohistochemical and biochemical studies (12, 15, 16, 17, 25) indicate that this reagent may prove to be valuable in the management of a broad scope of neoplasia. The demonstration of the drastic reduction in RB2/p130 expression, alterations in the genomic locus, and the growth suppression of the HONE-1 cell line upon restoration of functional RB2/p130 are, to our knowledge, the first genetic evidence to implicate that mutation or loss of function of RB2/p130 may be involved in tumor development (12). Additionally, pRb2/p130 was found to be an independent prognostic factor for endometrial carcinoma, and lack of pRb2/p130 expression could be used to identify individuals with at least a 5-fold increased risk of dying from the disease (25). These observations have been extended to lung cancer. Immunohistochemical studies of the expression patterns of the Rb family members (pRb/p105, p107, and pRb2/p130) in 235 specimens of lung cancer indicate that pRb2/p130 may play an important role for in the pathogenesis and progression of certain lung cancers (16, 17). In support of this hypothesis, the detection of a point mutation within a splice acceptor sequence eliminating exon 2 and extinguishing the production of detectable pRb2/p130 protein in a cell line of human small cell lung carcinoma was reported recently (41). Our identification,for the first time, of a high mutation rate of 78.5% in patient lung tumors further strengthens this notion. The presence of such pattern of identical mutations in the lung cancer samples examined raises many questions. One possibility is that this could be sort of a“fingerprint” of a smoking-related mutagen or others in the RB2/p130 gene. A paradigm is forming that the removal or inactivation of a functional pRb2/p130 protein by way of tumor viral oncoproteins [as is the case in SV40 large T antigen-associated mesothelioma (26)] or by way of genetic alteration in lung carcinoma may be a critical event in the malignant transformation of a cell. In consideration of these data, the impact of lung cancer in terms of morbidity and mortality on the Western world (18), and the results presented in this report, we suggest that RB2/p130 gene therapy may serve as an effective therapeutic alternative or adjuvant in combating lung cancer and is worth further intensive investigation. Additionally, identification of mutations within the RB2/p130 locus has possible implications on establishing the molecular diagnosis and/or prognosis of lung and other cancers that may be used to guide and to design standard or novel therapeutic regimes.

Our results in nude mice demonstrating that a single injection of retrovirus delivering the RB2/p130 ORF suppressed tumor growth in vivo on average by >92% and obtained complete regression in four of six tumors predict that RB2/p130 gene therapy may eventually become a viable therapeutic option for the management of lung and other cancers. RB2/p130 gene therapy was effective,even in the presence of a functional wild-type RB2/p130 protein product in H23 cells. Restoration of pRb2/p130 expression and function in the presence of a functionally inactivated pRb2/p130 protein in vivo had been demonstrated previously with a tetracycline-inducible expression system in a JC virus-transformed hamster glioblastoma model (35). Our results extend this concept to human neoplasms by using a gene delivery system with far more therapeutic potential and broaden the therapeutic range and implications of novel RB2/p130 gene therapeutics to not only include RB2/p130-deficient cells but also those retaining a wild-type allele.

The definition of a tumor suppressor gene requires that expression of its protein product inhibits tumor growth and that the gene is found mutated in primary tumors. We have demonstrated that RB2/p130 is a frequent target of mutational inactivation in human lung tumors and that ectopic expression of pRb2/p130 via a recombinant retrovirus can limit the proliferative potential of human lung tumor cells in vivo, thus fulfilling both of the criteria for this rigid definition. This is the critical genetic and functional evidence that RB2/p130 is a human tumor suppressor gene.

ACKNOWLEDGMENTS

We thank Dr. Hansjuerg Alder, Dr. Alfredo Ciccodicola, and Priya Hingorani for technical assistance in sequencing and confocal microscopy, Dr. K. Huebner (Thomas Jefferson University, Philadelphia,PA) for providing the H23 cell line, Dr. A. Kingsman (University of Oxford, Oxford, United Kingdom) for providing the HIT vectors, and Dr. R. Hawley (University of Toronto, Toronto, Ontario, Canada) for contributing the MSCV vectors.

Fig. 1.

A, SSCP analysis of exon 20 of the RB2/p130 gene with genomic DNA extracted from the tumor of patients 42 and 43 (42T and 43T). Genomic DNAs extracted from human normal placenta (Pla)and from matching peripheral blood lymphocytes (42N and 43N) were used as controls. B, SSCP analysis of exon 21 of the RB2/p130 gene with genomic DNA extracted from the tumor of patients 42 and 43 (42Tand 43T). Genomic DNAs extracted from human normal placenta (Pla) and from matching peripheral blood lymphocytes (42N and 43N) were used as controls. Arrow, abhorrent migratory bands. C, SSCP analysis of exon 21 of the RB2/p130 gene with genomic DNA extracted from the tumor of patients 46, 47, 48, 49, 50, 51, 52, 53, and 54 (46T, 47T,48T, 49T, 50T, 51T, 52T, 53T, and 54T). Genomic DNA extracted from human normal placenta (Pla) was used as control. D, SSCP analysis of exon 22 of the RB2/p130 gene with genomic DNA extracted from the tumor of patients 45, 47, 49, and 52 (45T, 47T, 49T, and 52T). Genomic DNAs extracted from human normal placenta(Pla) and from matching peripheral blood lymphocytes(45N, 47N, 49N, and 52N) were used as controls. Exon amplified and genomic DNA sources are identified above lanes.

Fig. 1.

A, SSCP analysis of exon 20 of the RB2/p130 gene with genomic DNA extracted from the tumor of patients 42 and 43 (42T and 43T). Genomic DNAs extracted from human normal placenta (Pla)and from matching peripheral blood lymphocytes (42N and 43N) were used as controls. B, SSCP analysis of exon 21 of the RB2/p130 gene with genomic DNA extracted from the tumor of patients 42 and 43 (42Tand 43T). Genomic DNAs extracted from human normal placenta (Pla) and from matching peripheral blood lymphocytes (42N and 43N) were used as controls. Arrow, abhorrent migratory bands. C, SSCP analysis of exon 21 of the RB2/p130 gene with genomic DNA extracted from the tumor of patients 46, 47, 48, 49, 50, 51, 52, 53, and 54 (46T, 47T,48T, 49T, 50T, 51T, 52T, 53T, and 54T). Genomic DNA extracted from human normal placenta (Pla) was used as control. D, SSCP analysis of exon 22 of the RB2/p130 gene with genomic DNA extracted from the tumor of patients 45, 47, 49, and 52 (45T, 47T, 49T, and 52T). Genomic DNAs extracted from human normal placenta(Pla) and from matching peripheral blood lymphocytes(45N, 47N, 49N, and 52N) were used as controls. Exon amplified and genomic DNA sources are identified above lanes.

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Fig. 2.

a, Southern blot of a 425-bp fragment of the puromycin resistance gene amplified from the MSCV Pac plasmid(Lane Pos.) or genomic DNA of A549 cells transduced with empty virus (Lane Neg.) or retroviruses carrying either the puromycin resistance gene alone (Lanes 1 and 3) or in combination with the RB2/p130gene (Lanes 2 and 4) at 4 or 8 days,respectively. The blot was probed with the same 425-bp fragment that was random primed-labeled with deoxycytidine 5′-[α-32P]triphosphate. b, colony assay of the H23 cell line transduced with 1 × 107 retroviruses carrying the puromycin resistance gene alone or in combination with the RB2/p130 gene. Supernatant collected from a cotransfection of 293T/17 cells with only the plasmids carrying gag/pol and env was used as negative control (plate Mock). c, Western blot analysis of total cell lysates from H23 cells transduced with retroviruses carrying the puromycin resistance or the neomycin resistance gene alone (Lanes 2 and 4) or in combination with the gene RB2/p130 (Lanes 3 and 5). H23 cells treated with supernatant collected from a cotransfection of 293T/17 cells with only the plasmids carrying gag/pol and env were used as control (Lane 1).

Fig. 2.

a, Southern blot of a 425-bp fragment of the puromycin resistance gene amplified from the MSCV Pac plasmid(Lane Pos.) or genomic DNA of A549 cells transduced with empty virus (Lane Neg.) or retroviruses carrying either the puromycin resistance gene alone (Lanes 1 and 3) or in combination with the RB2/p130gene (Lanes 2 and 4) at 4 or 8 days,respectively. The blot was probed with the same 425-bp fragment that was random primed-labeled with deoxycytidine 5′-[α-32P]triphosphate. b, colony assay of the H23 cell line transduced with 1 × 107 retroviruses carrying the puromycin resistance gene alone or in combination with the RB2/p130 gene. Supernatant collected from a cotransfection of 293T/17 cells with only the plasmids carrying gag/pol and env was used as negative control (plate Mock). c, Western blot analysis of total cell lysates from H23 cells transduced with retroviruses carrying the puromycin resistance or the neomycin resistance gene alone (Lanes 2 and 4) or in combination with the gene RB2/p130 (Lanes 3 and 5). H23 cells treated with supernatant collected from a cotransfection of 293T/17 cells with only the plasmids carrying gag/pol and env were used as control (Lane 1).

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Fig. 3.

Tumor growth in nude mice injected with 2.5 × 106 H23 cells and the average tumor volume over time of studies 1 and 2. At day 15, tumors were transduced with an intratumoral injection (20 μl) of a solution containing 5 × 106 retroviruses carrying the puromycin resistance gene alone (a and b), the lacZ gene (c and d), or the RB2/p130 gene (e and f).

Fig. 3.

Tumor growth in nude mice injected with 2.5 × 106 H23 cells and the average tumor volume over time of studies 1 and 2. At day 15, tumors were transduced with an intratumoral injection (20 μl) of a solution containing 5 × 106 retroviruses carrying the puromycin resistance gene alone (a and b), the lacZ gene (c and d), or the RB2/p130 gene (e and f).

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Fig. 4.

a, H&E staining of an H23 tumor grown in nude mice transduced with the retroviruses carrying the puromycin resistance gene (Pac) showing the typical features of an adenocarcinoma poorly differentiated. ×1000. b,immunohistochemistry with rabbit polyclonal anti-pRb2/p130 as primary antibody, polyclonal goat antirabbit biotinylated antibody as secondary antibody, and diaminobenzidine as chromogen; hematoxylin counterstaining shows high levels of protein expression (≥75%positive cells) in the nuclei of H23 tumor cells transduced with RB2/p130 retroviruses. ×1000. c, Western blot analysis of protein lysates from H23 tumors in nude mice. High expression levels of pRb2/p130 were found in two different tumors upon transduction with a retrovirus carrying the RB2/p130 gene (Lanes 1 and 2) compared with that of protein lysates from tumors transduced with retroviruses carrying the puromycin resistance gene alone (Lane 3) or the lacZ gene (Lane 4). PRINS is shown, using primers that amplify a 425-nucleotide fragment of the puromycin resistance gene present in the Pac, lacZ,and RB2/p130 retroviruses in OTC frozen sections. d,tumor samples from H23 tumors transduced with empty virus as a negative control. e, tumor samples from H23 tumors transduced with retroviruses carrying the puromycin resistance gene alone. f, tumor samples from H23 tumors transduced with retrovirus carrying the RB2/p130 gene. x10 mm bar, 10 μm.

Fig. 4.

a, H&E staining of an H23 tumor grown in nude mice transduced with the retroviruses carrying the puromycin resistance gene (Pac) showing the typical features of an adenocarcinoma poorly differentiated. ×1000. b,immunohistochemistry with rabbit polyclonal anti-pRb2/p130 as primary antibody, polyclonal goat antirabbit biotinylated antibody as secondary antibody, and diaminobenzidine as chromogen; hematoxylin counterstaining shows high levels of protein expression (≥75%positive cells) in the nuclei of H23 tumor cells transduced with RB2/p130 retroviruses. ×1000. c, Western blot analysis of protein lysates from H23 tumors in nude mice. High expression levels of pRb2/p130 were found in two different tumors upon transduction with a retrovirus carrying the RB2/p130 gene (Lanes 1 and 2) compared with that of protein lysates from tumors transduced with retroviruses carrying the puromycin resistance gene alone (Lane 3) or the lacZ gene (Lane 4). PRINS is shown, using primers that amplify a 425-nucleotide fragment of the puromycin resistance gene present in the Pac, lacZ,and RB2/p130 retroviruses in OTC frozen sections. d,tumor samples from H23 tumors transduced with empty virus as a negative control. e, tumor samples from H23 tumors transduced with retroviruses carrying the puromycin resistance gene alone. f, tumor samples from H23 tumors transduced with retrovirus carrying the RB2/p130 gene. x10 mm bar, 10 μm.

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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by NIH Grants RO1 CA 60999-01A1, PO1 NS 36466, Sbarro Institute for Cancer Research and Molecular Medicine(to A. G.), and by 40% and 60% Ministero dell’Universitá e Ricerca Scientifica e Tecnologica grants to University of Bologna. P. P. C. is the recipient of a fellowship from the Associazione Leonardo di Capua, Napoli, Italy.

3

The abbreviations are: Rb, retinoblastoma; CMV,cytomegalovirus; MLV, Moloney leukemia virus; MSCV, murine stem cell virus; SSCP, single strand conformation polymorphism; PRINS,primer in situ DNA synthesis; ORF, open reading frame;LTR, long terminal repeat; CI, confidence interval.

4

P. P. Claudio, C. Cinti, and A. Giordano,unpublished results.

Table 1

Mutations in 11 primary lung carcinoma tumors determined by SSCP and DNA sequence analysis

Patient (tumor type)Mutated base is underlinedZygosityEffectBasepair no(s).Codon no.Exon no.
40 (poorly differentiated adenocarcinoma) AGA → AGT-A Homozygous Insertion 3320/3321 1084 22 
 AGA → AGC-A Homozygous Insertion 3326/3327 1086 22 
 AAA → ATA-A Homozygous Insertion 3367/3368 1100 22 
41 (moderately differentiated adenocarcinoma) AGA → AGT-A Homozygous Insertion 3320/3321 1084 22 
 AGA → AGC-A Homozygous Insertion 3326/3327 1086 22 
42 (poorly differentiated adenocarcinoma) CGA → GGA Heterozygous Arg → Gly 3277 1070 21 
 AAC → TTHeterozygous Asn → Phe 3304; 3305 1079 21 
 AAA → ATA-A Homozygous Insertion 3367/3368 1100 22 
43 (poorly differentiated squamous cell carcinoma) CAG → TAG Heterozygous Stop codon 3214 1049 21 
 CGA → GGA Heterozygous Arg → Gly 3277 1070 21 
 AGA → AGTHomozygous Insertion 3320/3321 1084 22 
 AGA → AGC-A Homozygous Insertion 3326/3327 1086 22 
45 (small cell lung cancer) GTG → GGHeterozygous 5′ donor   21 
46 (poorly differentiated adenocarcinoma) AAG → AGHeterozygous Lys → Arg 3317 1083 21 
 GTG → GGHeterozygous 5′ donor   21 
47 (poorly differentiated adenocarcinoma) GTG → GGHeterozygous 5′ donor   21 
49 (moderately differentiated adenocarcinoma) AAG → AGHeterozygous Lys → Arg 3317 1083 21 
 GTG → GGHeterozygous 5′ donor   21 
50 (poorly differentiated adenocarcinoma) AAG → AGHeterozygous Lys → Arg 3317 1083 21 
 GTG → GGHeterozygous 5′ donor   21 
51 (poorly differentiated adenocarcinoma) AAC → TTHeterozygous Asn → Phe 3304; 3305 1079 21 
 AAG → ACHeterozygous Lys → Thr 3317 1083 21 
 GTG → GGHeterozygous 5′ donor   21 
52 (poorly differentiated adenocarcinoma) AAG → AGHeterozygous Lys → Arg 3317 1083 21 
 GTG → GGHeterozygous 5′ donor   21 
Patient (tumor type)Mutated base is underlinedZygosityEffectBasepair no(s).Codon no.Exon no.
40 (poorly differentiated adenocarcinoma) AGA → AGT-A Homozygous Insertion 3320/3321 1084 22 
 AGA → AGC-A Homozygous Insertion 3326/3327 1086 22 
 AAA → ATA-A Homozygous Insertion 3367/3368 1100 22 
41 (moderately differentiated adenocarcinoma) AGA → AGT-A Homozygous Insertion 3320/3321 1084 22 
 AGA → AGC-A Homozygous Insertion 3326/3327 1086 22 
42 (poorly differentiated adenocarcinoma) CGA → GGA Heterozygous Arg → Gly 3277 1070 21 
 AAC → TTHeterozygous Asn → Phe 3304; 3305 1079 21 
 AAA → ATA-A Homozygous Insertion 3367/3368 1100 22 
43 (poorly differentiated squamous cell carcinoma) CAG → TAG Heterozygous Stop codon 3214 1049 21 
 CGA → GGA Heterozygous Arg → Gly 3277 1070 21 
 AGA → AGTHomozygous Insertion 3320/3321 1084 22 
 AGA → AGC-A Homozygous Insertion 3326/3327 1086 22 
45 (small cell lung cancer) GTG → GGHeterozygous 5′ donor   21 
46 (poorly differentiated adenocarcinoma) AAG → AGHeterozygous Lys → Arg 3317 1083 21 
 GTG → GGHeterozygous 5′ donor   21 
47 (poorly differentiated adenocarcinoma) GTG → GGHeterozygous 5′ donor   21 
49 (moderately differentiated adenocarcinoma) AAG → AGHeterozygous Lys → Arg 3317 1083 21 
 GTG → GGHeterozygous 5′ donor   21 
50 (poorly differentiated adenocarcinoma) AAG → AGHeterozygous Lys → Arg 3317 1083 21 
 GTG → GGHeterozygous 5′ donor   21 
51 (poorly differentiated adenocarcinoma) AAC → TTHeterozygous Asn → Phe 3304; 3305 1079 21 
 AAG → ACHeterozygous Lys → Thr 3317 1083 21 
 GTG → GGHeterozygous 5′ donor   21 
52 (poorly differentiated adenocarcinoma) AAG → AGHeterozygous Lys → Arg 3317 1083 21 
 GTG → GGHeterozygous 5′ donor   21 
Table 2

Soft-agar colony formation by H23

Cells were seeded at an initial density of 5 × 103 cells into 60-mm six-well culture dishes in duplicate in 0.3% agar. Colonies containing at least 50 cells were scored after 23 days of growth. Total colony count from six dishes is shown.

Soft Agar Colony Formation Assay
H23 MockH23 MSCV PacH23 MSCV Rb2/p130
Experiment 1 1125 108a 
Experiment 2 1546 165b 
Soft Agar Colony Formation Assay
H23 MockH23 MSCV PacH23 MSCV Rb2/p130
Experiment 1 1125 108a 
Experiment 2 1546 165b 
a

Decrease in number of colonies compared with H23 MSCV Pac is 90.4%; the fold reduction for the total number of colonies in soft agar in experiment 1 is 10.4.

b

Decrease in number of colonies compared with H23 MSCV Pac is 89.4%; the fold reduction for the total number of colonies in soft agar in experiment 2 is 9.36.

Table 3

RB2/p130 ex vivo transduction and tumor formation of H23 cells in nude mice

Average tumor volumes, mm95% CI, tumor volumesAverage weight of tumors, g95% CI, tumor weight
Pac experiment 1     
Day 0 ± 0   
Day 15 28.358 ± 24.92   
Day 30 364.583 ± 291.72 0.216 ± 0.081 
Pac experiment 2     
Day 0 —   
Day 7 —   
Day 16 281.666 ± 189.65   
Day 20 1056.666 ± 421.90 0.288 ± 0.244 
Rb2/p130 experiment 1     
Day 0 —   
Day 15 0.833 ± 0.485   
Day 30 10 ± 6.366 0.012 ± 0.004 
Rb2/p130 experiment 2     
Day 0 —   
Day 7 —   
Day 16 5.416 ± 2.658   
Day 20 28.083 ± 18.282 0.067 ± 0.020 
Average tumor volumes, mm95% CI, tumor volumesAverage weight of tumors, g95% CI, tumor weight
Pac experiment 1     
Day 0 ± 0   
Day 15 28.358 ± 24.92   
Day 30 364.583 ± 291.72 0.216 ± 0.081 
Pac experiment 2     
Day 0 —   
Day 7 —   
Day 16 281.666 ± 189.65   
Day 20 1056.666 ± 421.90 0.288 ± 0.244 
Rb2/p130 experiment 1     
Day 0 —   
Day 15 0.833 ± 0.485   
Day 30 10 ± 6.366 0.012 ± 0.004 
Rb2/p130 experiment 2     
Day 0 —   
Day 7 —   
Day 16 5.416 ± 2.658   
Day 20 28.083 ± 18.282 0.067 ± 0.020 
Table 4

RB2/p130 in vivo transduction and tumor regression of H23 cells in nude mice

Average tumor volumes, mm95% CI, tumor volumesAverage weight of tumors, g95% CI, tumor weight
Pac experiment 1     
Day 0 —   
Day 10 17.333 ± 3.344   
Day 15 23.666 ± 4.276   
Day 18 46.208 ± 17.262   
Day 20 46.375 ± 17.100   
Day 22 64.50 ± 17.852   
Day 27 122.583 ± 35.174   
Day 31 218.833 ± 93.181 0.245 ± 0.078 
Pac experiment 2     
Day 0 —   
Day 10 14 ± 4.747   
Day 15 19.166 ± 4.223   
Day 18 36.041 ± 15.530   
Day 20 39.958 ± 12.650   
Day 22 58.583 ± 14.589   
Day 27 111.166 ± 32.860   
Day 31 204.333 ± 70.547 0.249 ± 0.105 
LacZ experiment 1     
Day 0 —   
Day 10 21.50 ± 0.946   
Day 15 23.583 ± 2.435   
Day 18 44.850 ± 12.533   
Day 20 47.416 ± 13.148   
Day 22 66.250 ± 19.398   
Day 27 134.333 ± 74.222   
Day 31 342.583 ± 228.184 0.300 ± 0.096 
LacZ experiment 2     
Day 0 —   
Day 10 19.666 ± 4.488   
Day 15 23 ± 3.379   
Day 18 43.833 ± 11.297   
Day 24 58.333 ± 14.567   
Day 26 81 ± 14.614   
Day 28 127.666 ± 11.186   
Day 31 249 ± 53.937 0.309 ± .075 
Rb2/p130 experiment 1     
Day 0 —   
Day 10 18.833 ± 2.382   
Day 15 20.416 ± 2.272   
Day 18 11.483 ± 3.991   
Day 24 9.650 ± 3.950   
Day 26 7.983 ± 3.659   
Day 28 5.166 ± 2.171   
Day 31 3.150 ± 1.662 0.020 ± 0.024 
Rb2/p130 experiment 2     
Day 0 —   
Day 10 21.5 ± 3.067   
Day 15 24.583 ± 2.538   
Day 18 19.333 ± 4.738   
Day 24 14.1 ± 5.028   
Day 26 11.833 ± 5.530   
Day 28 9.5 ± 5.104   
Day 31 7.1 ± 4.331 0.042 ± 0.027 
Average tumor volumes, mm95% CI, tumor volumesAverage weight of tumors, g95% CI, tumor weight
Pac experiment 1     
Day 0 —   
Day 10 17.333 ± 3.344   
Day 15 23.666 ± 4.276   
Day 18 46.208 ± 17.262   
Day 20 46.375 ± 17.100   
Day 22 64.50 ± 17.852   
Day 27 122.583 ± 35.174   
Day 31 218.833 ± 93.181 0.245 ± 0.078 
Pac experiment 2     
Day 0 —   
Day 10 14 ± 4.747   
Day 15 19.166 ± 4.223   
Day 18 36.041 ± 15.530   
Day 20 39.958 ± 12.650   
Day 22 58.583 ± 14.589   
Day 27 111.166 ± 32.860   
Day 31 204.333 ± 70.547 0.249 ± 0.105 
LacZ experiment 1     
Day 0 —   
Day 10 21.50 ± 0.946   
Day 15 23.583 ± 2.435   
Day 18 44.850 ± 12.533   
Day 20 47.416 ± 13.148   
Day 22 66.250 ± 19.398   
Day 27 134.333 ± 74.222   
Day 31 342.583 ± 228.184 0.300 ± 0.096 
LacZ experiment 2     
Day 0 —   
Day 10 19.666 ± 4.488   
Day 15 23 ± 3.379   
Day 18 43.833 ± 11.297   
Day 24 58.333 ± 14.567   
Day 26 81 ± 14.614   
Day 28 127.666 ± 11.186   
Day 31 249 ± 53.937 0.309 ± .075 
Rb2/p130 experiment 1     
Day 0 —   
Day 10 18.833 ± 2.382   
Day 15 20.416 ± 2.272   
Day 18 11.483 ± 3.991   
Day 24 9.650 ± 3.950   
Day 26 7.983 ± 3.659   
Day 28 5.166 ± 2.171   
Day 31 3.150 ± 1.662 0.020 ± 0.024 
Rb2/p130 experiment 2     
Day 0 —   
Day 10 21.5 ± 3.067   
Day 15 24.583 ± 2.538   
Day 18 19.333 ± 4.738   
Day 24 14.1 ± 5.028   
Day 26 11.833 ± 5.530   
Day 28 9.5 ± 5.104   
Day 31 7.1 ± 4.331 0.042 ± 0.027 
1
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