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Molecular Biology and Genetics |
Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107 [P. P. C., C. M. H., C. P., G. R, C. M., A. G.]; Sbarro Institute for Cancer Research and Molecular Medicine, Philadelphia, Pennsylvania 19107 [P. P. C., C. M. H., C. P., A. G.]; Dipartimento di Scienze Odontostomatologiche e Maxillo-Facciali, University of Naples "Federico II," 80100 Napoli, Italy [P. P. C.]; Istituto di Citomorfologia Normale e Patologica, Consiglio Nazionale delle Ricerche, c/o Istituti Ortopedici Rizzoli, 40136 Bologna, Italy [C. C., N. M. M.]; Laboratorio di Biologia Cellulare e Microscopia elettronica, Istituti di Ricerca "Codivilla Putti," IOR, 40100 Bologna, Italy [N. M. M.]; Hamon Center for Ther. Onc. Res., University of Texas Southwestern Medical Center, Dallas, Texas 75235-8593 [J. D. M.]; Bristol-Meyers Squibb, Princeton, New Jersey 08543-4000 [L. G.]; Institute di Anatomia e Istologia Patologica, Universita di Siena, 53100 Siena, Italy [L. L.]; Dipartimento di Scienze Oftalmologiche e Neurochirurgiche, Universita’ di Siena, 53100 Siena, Italy [G. M. T.]; Azienda Ospedaliera Cotugno, Servizio di Anatomia, Istologia e Citologia Patologica, Napoli, Italy [P. M.]; Istituto di Malattie dell’Apparato Respiratorio, II Universita’ degli Studi di Napoli, Istituto di Ricerca Cardio-Pneumologico A. O. "Monaldi," Napoli, Italy [M. C.]; and Istituto di Anatomia Patologica, Facolta’ di Medicina, II Universita’ di Napoli, Napoli 80122, Italy [G. G. G.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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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 mM
L-glutamine. The 293T/17 cell line was maintained in DMEM
supplemented with 10% heat-inactivated fetal bovine serum and 2
mM L-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'-[
-[35S]thio]triphosphate, 2
units of high-fidelity Taq polymerase (Boehringer Mannheim,
Indianapolis, IN), and primers at a final concentration of 0.4
µM. 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 x 104
cells/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'[-[32P]triphosphate (DuPont NEN Boston,
MA) and used as probe. The membrane was washed with 0.2% SDS and 2x
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 x 105 cells/dish and transduced with 20
µl, 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 2x 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 x 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 x 103
cells) 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 x 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 x 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 x 106 H23 cells/flank. After 15 days when
the tumors reached a volume of 20 mm3, each
tumor was transduced with 5 x 106
retroviruses carrying the Pac gene alone or the
Pac gene and the E. coli
ß-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) x (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 t
test 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 ß-Galactosidase Assay.
Immumohistochemistry was performed with a rabbit polyclonal immune
serum anti-COOH-terminal pRb2/p130 (16
, 17)
or the
anti-ß-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 ß-galactosidase assay was performed essentially as described
previously (28)
.
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RESULTS |
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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.
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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-G1
growth-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 x 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. 2a
). 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.
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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. 2a
, 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
[-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. 2a
).
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. 2b
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. 2b
) 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. 2c
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)
.
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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)
.
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). 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).
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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 vivo
transduction 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. 4a
(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. 4b
). ß-Galactosidase
activity and immunohistochemistry used to demonstrate the expression of
a functional ß-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 lacZ
retroviruses (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. 4c
and
data not shown).
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In essence, we have produced an effective system for gene delivery and stable integration of the RB2/p130 gene in vitro and 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.
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FOOTNOTES |
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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. 
2 To whom requests for reprints should be
addressed, at Pathology, Anatomy, and Cell Biology, Kimmel Cancer
Institute, Thomas Jefferson University, 1020 Locust Street, Room 226,
Philadelphia, PA 19107. Phone: (215) 503-0781; Fax: (215) 923-9626;
E-mail: agiordan{at}lac.jci.tju.edu
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.
Received 6/ 3/99. Accepted 11/11/99.
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75%
positive cells) in the nuclei of H23 tumor cells transduced with
RB2/p130 retroviruses. x1000. 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.