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Molecular Biology and Genetics |
Istituto di Citomorfologia Normale e Patologica, CNR, 40136 Bologna, Italy [C. C., L. M. N., N. M. M.]; Departments of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [P. P. C., C. M. H., Y. F., A. G.]; Dipartimento di Scienze Odontostomatologiche e Maxillo-Facciali, Università di Napoli "Federico II," 80100 Naples, Italy [P. P. C.]; Istituto di Anatomia Umana Normale, Università di Ferrara, 44100 Ferrara, Italy [L. M. N.]; Laboratorio di Biologia Cellulare e Microscopia Elettronica, Istituti di Ricerca "Codivilla Putti," IOR, 40100 Bologna, Italy [N. M. M.]; and Istituto di Anatomia e Istologia Patologica [L. L.] and Dipartimento di Scienze Oftalmologiche e Neurochirurgiche [G. M. T.], Università di Siena, 53100 Siena, Italy
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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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In this report, we describe a prevailing nuclear exclusion of pRb2/p130 in the lymphoid tumor cell lines that is dependent on the presence of mutations that affect the NLS of pRb2/p130. Neither abhorrent cytoplasmic localization nor NLS-specific mutations were found in the osteosarcoma cell lines.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Saos-2 cells were plated at a concentration of 1 x 106 cells/plate in triplicate. After 24 h, the cells were transfected by the standard calcium phosphate precipitation method (25) .
Immunofluorescence and Confocal Microscopy Analysis.
All cell lines were fixed in 4% paraformaldehyde in 1x PBS and
permeabilized with 0.8% Triton X-100 for 15 min. Slides were incubated
to block nonspecific binding with 2% BSA and 3% normal goat serum in
PBS (immunoreaction buffer) for 30 min at 37°C, reacted with the
primary monoclonal anti-Rb2/p130 antibody (clone 10; Transduction
Laboratories, Lexington, KY) diluted in immunoreaction buffer 1:50 for
3 h at 37°C, and then reacted with secondary FITC-conjugated
rabbit antimouse IgG (Sigma, St. Louis, MO) for 1 h at 37°C. DNA
was counterstained with 4',6-diamidino-2-phenylindole (Sigma) to assess
the nuclear domains. The samples were analyzed by a Zeiss LSM410 (Carl
Zeiss) confocal laser scanning microscope equipped with a 100x oil
immersion lens (numerical aperature = 1.4) and a
488/514 nm argon laser. Image acquisition, recording, and filtering
were performed on the z series of confocal data (stacks) by
an Indy 4600 graphic workstation (Silicon Graphics) as described
previously (31)
.
Western Blot Analysis.
Whole cell lysates were prepared by resuspending cell pellets in 200
µl of lysis buffer (50 mM Tris-HCl, 5 mM
EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton, 0.1
mM Na3VO4, and
protease inhibitors 1 mM phenylmethylsulfonyl fluoride and
1 µg/ml aprotinin and leupeptin). The lysates were cleared by
centrifugation for 15 min at 13,000 x g at
4°C, and total protein extracts were determined (25)
.
The protein (40 µg) was denatured by boiling in 2x Laemmli buffer
[62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10%
glycerol, 5% ß-mercaptoethanol, and bromphenol blue] and
size-fractionated by electrophoresis in 6% SDS-polyacrylamide gel. The
electrophoretic transfer of the protein to a polyvinylidene difluoride
membrane (Millipore) was performed in
3-(cyclohexylamino)propanesulfonic acid buffer [10
mM 3-(cyclohexylamino)propanesulfonic acid and
2% methanol (pH 11)]. The membrane was blocked with 5% fat-free
dried milk in TBS-T buffer [2 mM Tris, 13.7
mM NaCl, and 0.1% Tween-20 (pH 7.6)] and
incubated with the primary monoclonal antibody (Transduction
Laboratories) at a dilution of 1:500 in 3% milk. After several washes,
the membrane was incubated with antimouse antibody coupled with
horseradish peroxidase (Amersham) for 1 h and detected using the
enhanced chemiluminescence system (Dupont NEN; Ref. 32
).
PCR and SSCP Analysis.
The PCR reaction mixture (50 µl) contained genomic DNA at a final
concentration of 4 ng/µl, 0.2 mM of each of the four
deoxynucleotide triphosphates, 2 units of Klen TaqI (Ab
Peptides), and the intron primer panels (exon 19,
5'-AGGTCCTATCACCAAGGGTGT-3'; exon 19 reverse primer,
5'-GCTTAGTTACTTCTTCAAGGC-3'; exon 20,
5'-GAGAAAGTTAATATCCTAGCTG-3'; exon 20 reverse primer,
5'-GTGAATGGTCCATATATAAATCA-3'; exon 21,
5'-TGGTTTAGCACACCTCTTCAC-3'; exon 21 reverse primer,
5'-GCTTAGCACAAACCCTGTTTC-3'; exon 22, 5'-CTGAGCTATGTGCATTTGCA-3'; exon
22 reverse primer, 5'-AAGGCTGCTGCTAAACAGAT-3') at a final concentration
of 0.4 µM each. Thirty-five cycles of
denaturation (95°C, 1 min), annealing (55°C, 1 min), and extension
(72°C, 1 min) linked to one cycle at 72°C for 7 min were carried
out in a thermal cycler (Perkin-Elmer, Norwalk, CT). For all of the
tumor cell lines, reverse transcription-PCR was performed using primers
5'-ATTTAGCAGCTGTCCGC-3' (forward) and 5'-AAGGCTGCTGCTAAACAGAT-3'
(reverse) that amplified the region from nucleotide 2603–3540. The
annealing temperature used was 56°C, and the cDNAs amplified were 937 bp.
MDE gel solution (FMC BioProducts, Rockland, ME; supplied as 2x liquid concentrate) was used for SSCP analysis. For a 100-ml total volume, 25 ml of 2x MDE gel solution, 6 ml of 10x TBE buffer, 69 ml of deionized water, 40 µl of N,N,N',N'-tetramethylethylenediamine, and 400 µl of 10% ammonium persulfate were used as recommended by AT Biochem. Genomic PCR products (2 µl) were added to 9 µl of stop solution and heated to 94°C for 2 min. The denatured DNA was placed directly on ice for several minutes and then run through the MDE gel at 8 W constant power for 8 h at 15°C in 0.6x TBE running buffer. The bands were visualized with a silver staining method (Bio-Rad, Hercules, CA).
Sequence Analysis.
The PCR products of genomic DNAs and cDNAs were resolved on a 1.5%
ethidium bromide-stained agarose gel. Bands were cut from the gels. DNA
was purified using the QUIAquick gel extraction kit (Qiagen, Santa
Clarita, CA) and used for automated DNA forward and reverse sequencing
using dideoxy terminator reaction chemistry for sequence analysis on
the Applied Biosystem Model 373A DNA sequencer. The forward and reverse
sequences were repeated more times for each PCR product.
Site-directed in Vitro Mutagenesis.
Oligonucleotides encoding the putative bipartite NLS of pRb2/p130, the
region encoding amino acids 1082–1102, were synthesized with the
addition of an ATG transcriptional start site at the
NH2 terminus, 5' HindIII and 3'
BamHI restriction sites, and two adenosine residues just
before the BamHI site to keep the heterologous fusion
protein between the bipartite NLS and the NH2
terminus of the EGFP protein in frame. The oligonucleotides were
annealed and ligated into the HindIII and BamHI
restriction sites of the pEGFP-N1 expression construct to form the
pEGFP-N1-NLS construct. pEGFP-N1-NLS-NQ1, pEGFP-N1-NLS-NQ2, and
pEGFP-N1-NLS-NQ1&2 were constructed as described previously, except
that the oligonucleotides synthesized encoded point mutations that
altered amino acids Lys1082 to Asn and
Arg1083 to Gln in the first bipartite site and
Lys1100 to Asn and Arg1102
to Gln in the second site, and the Lys-to-Asn and Arg-to-Gln mutations
were combined in both sites, respectively.
The point mutations in the bipartite NLS of full-length Rb2/p130 cDNA were formed by an extra long PCR technique developed in our laboratory that allows site-specific mutagenesis (33) . Wild-type Rb2/p130 cDNA with an exogenous HA epitope at the COOH terminus (pcDNA3-Rb2/p130-HA) as well as with the HA epitope and the c-myc major NLS (HAN) at the COOH terminus (pcDNA3-Rb2/p130-HAN) in the pcDNA3 vector were used as the PCR template. The pcDNA3-Rb2/p130- constructs contained the equivalent Lys-to-Asn and Arg-to-Gln mutations in the bipartite NLS as described above in the first site (NLS-NQ1), the second site (NLS-NQ2), or both (NLS-NQ1&2), with either the HA or HAN epitope, as indicated. The pcDNA3-Rb2-p130-WT and pCMV-CD20 constructs, which express the wild-type pRb2/p130 without any epitope tags and the interleukin 2 receptor, respectively, were described previously (25) .
Flow Cytometry Analysis and Colony Formation Assays.
Flow cytometry analysis (FACS) was carried out according to the
procedure described previously (25)
. Ten µg of DNA
(pcDNA3, pcDNA3-Rb2/p130-WT, pcDNA3-Rb2/p130-HA, pcDNA3-Rb2/p130-HAN,
pcDNA3-Rb2/p130-NLS-NQ1-HA, pcDNA3-Rb2/p130-NLS-NQ1-HAN,
pcDNA3-Rb2/p130-NLS-NQ2-HA, pcDNA3-Rb2/p130-NLS-NQ2-HAN,
pcDNA3-Rb2/p130-NLS-NQ1&2-HA, or pcDNA3-Rb2/p130-NLS-NQ1&2-HAN)
were cotransfected with 2 µg of pCMV-CD20. Eighteen h after
transfection, the cells were washed twice with 1x PBS and once with
culture medium and incubated with fresh medium at 37°C. The cells
were collected with 1x PBS and 0.1% EDTA at 48 h, processed for
FACS analysis by incubation with the FITC-conjugated anti-CD20
monoclonal antibody (Becton Dickinson, Franklin Lakes, NJ), fixed in
70% ethanol, stained with propidium iodide, and treated with RNase A,
as described previously (25)
. FACS analysis was performed
on a Coulter Elite apparatus, and data from 1 x 104 CD20-positive cells were used to determine
the cell cycle distribution of the selected cells.
The colony formation assay was performed by transfecting triplicate dishes of Saos-2 cells with 10 µg of the indicated DNAs according to the procedure described previously (25) . Saos-2 cells were selected with 800 µg/ml G418 for 3 weeks and then stained with 1% methylene blue in 50% ethanol.
Detection of EGFP and EGFP-Fusion Proteins.
Sterile glass coverslips were placed into 10-cm tissue culture dishes,
and Saos-2 cells were plated 1 x 106 cells/dish. Forty-eight h after transfection
with the indicated pEGFP-N1 (Clontech, Palo Alto, CA) constructs and
fusion proteins, cells were washed three times with 1x PBS and fixed
directly on coverslips in freshly made 4% paraformaldehyde for 30 min
at room temperature. Cells were then washed twice with 1x PBS,
counterstained with 0.04 mg/ml propidium iodide in 1x PBS, mounted
onto a glass microscope slide with a drop of PBS, and sealed with
rubber cement. Slides were examined with a Leitz Wetzlar
fluorescence-equipped microscope. Images for illustration purposes were
obtained using a cooled charge-coupled device camera (Princeton
Instruments, Trenton, NJ). The images of the EGFP and EGFP-fusion
proteins were superimposed on those of the propidium iodide staining.
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RESULTS |
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As depicted in Fig. 1
, all of the lymphoid tumor cell lines show a prevailing localization of
the protein at the cytoplasmic level (Fig. 1, a–d
), whereas
normal human lymphocytes (Fig. 1e
) used as a control and the
osteosarcoma cell lines (Fig. 1, f–i
) all exhibit an
exclusive nuclear localization of pRb2/p130.
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Mutational Screening.
In pRb2/p130, like pRb/p105 (12)
, a putative NLS
consisting of two clusters of basic residues separated by a stretch of
amino acids is present in the COOH terminus of the protein. This
sequence is highly homologous with the pRb/p105 NLS (Table 1)
(in bold). In addition, the majority of the point mutations in
RB/p105 are located in the B domain and COOH-terminal region
(34)
. To verify whether or not the intracellular
distribution of the pRb2/p130 protein depends on mutations that affect
NLS motifs differently, we studied the structure of exons 19–22 of the
RB2/p130 gene that encode the B domain and the COOH terminus
of the protein, where the putative NLS is located
(30)
. Genomic DNA sequences from coding exons 19–22 were
amplified and screened for mutation by SSCP analysis. The direct PCR
products were sequenced to identify the actual mutations. To reduce
artifactual misincorporation generated by Taq polymerase, we used
high-fidelity Taq, and forward and reverse sequences were repeated
three times for each sample using PCR products derived from different
amplifications. Human placental DNA and DNA from normal peripheral
blood lymphocytes were used as controls for PCR reactions, SSCP
analysis, and sequences. CCRF-CEM cells showed a homozygous insertion
in exon 21, and Daudi and Molt-4 cell lines showed a heterozygous
insertion that caused the loss of the bipartite NLS resulting from a
shift in the coding frame with a consequent stop codon upstream of the
NLS present in exon 22 (Table 2)
. The Jurkat cell line showed two heterozygous mutations located at the
end of exon 21 that caused the loss of the two serines upstream of the
NLS. Moreover, two other heterozygous mutations inside the first and
second regions of the bipartite NLS were present in this cell line
(Table 2)
. These compound mutations caused the loss of nuclear
localization of the protein as shown by immunocytochemistry (Fig. 1
).
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.
To rule out the hypothesis that mutations of the RB2/p130
gene are a product of cell growth in culture, we performed the same
genetic analysis on primary Burkitt’s lymphomas positive for EBV. The
mutations and insertions present in four primary tumors were the same
as those found in the Daudi cell line, which is derived from a
Burkitt’s lymphoma (Table 2)
.
Because some of the mutations found in the lymphoid cell lines caused a premature stop codon, predicting a shorter trascription product with respect to wild type, we performed a Western blot analysis to detect the molecular weight of the pRb2/p130 protein in these cell lines.
Western Blot Analysis.
Western blot analysis of the cell lysates revealed the wild-type
phosphorylated and hypophosphorylated forms
(Mr
130,000/Mr 120,000) of the
protein in the osteosarcoma cell lines (Saos and Hos) and in three of
the lymphoid cell lines examined (Jurkat, Daudi, and Molt-4). One band
at a different molecular weight with respect to the wild type is
detectable in the same lymphoid cell lines (Fig. 2
). In the Jurkat cell line, only the unphosphorylated form of
Mr 120,000 was present because two
heterozygous mutations upstream of the NLS changed two serines into two
arginines, altering two putative sites of phosphorylation (see
Table 2
). Therefore, mutations resulting in a persistent
hypophosphorylated form of the protein should result in an enhanced
growth suppressive activity, but the concomitant presence in this cell
line of two point mutations in the bipartite NLS prevents this
activity, confining the protein to the cytoplasm. An abnormal band at
Mr 116,000 was present in CCRF-CEM,
Daudi, and Molt-4 cells, resulting from a guanosine or cytosine
insertion, respectively, that caused a frameshift in the sequence and a
premature stop codon with a protein of predicted molecular weight of
116,000. In addition, the presence of the
Mr
130,000/Mr 116,000 and
Mr
130,000/Mr
120,000/Mr 116,000 bands in Daudi and
Molt-4 cells, respectively, was the result of different heterozygous
mutations in these cell lines that disrupt the phosphorylation status
in a different way (see Table 2
). In fact, these mutations also changed
the amount of serines and threonines that could be phosphorylated.
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). Point mutations were also constructed in the first region
of the putative bipartite NLS, resulting in a change of amino acids
Lys1082 to Asn and Arg1083
to Gln (pEGFP-N1-NLS-NQ1), and in the second site, resulting in a
change of amino acids Lys1100 to Asn and
Arg1102 to Gln (pEGFP-N1-NLS-NQ2), as well as the
combination mutations in both bipartite sites
(pEGFP-N1-NLS-NQ1&2). This was done because similar point mutations in
the downstream sequences of the bipartite NLS of pRb/p105
(12)
and nucleoplasmin disrupted nuclear localization
(35)
. The plasmids were transfected into Saos-2 cells, and
the locations of the ectopically expressed proteins were determined by
fluorescence microscopy. Saos-2 cells were chosen because pRb2/p130 is
found to be exclusively nuclear in these cells, confirming that they do
not harbor any mutations in other proteins that may affect nuclear
shuttling directed by this region. The cells were counterstained with
propidium iodide. As shown in Fig. 3
, the level of background green
fluorescence was determined to be nonsignificant in mock-transfected
cells (Fig. 3A
). In pEGFP-N1-transfected cells, EGFP was
expressed ubiquitously in the cell (Fig. 3B
). Fusion of the
wild-type putative bipartite NLS to EGFP localized expression
exclusively in the nucleus (Fig. 3C
). EGFP fused to point
mutations in either the upstream region (Fig. 3D
) or
downstream region of the bipartite NLS (Fig. 3E
) resulted in
primarily nuclear expression. Mutations in both bipartite sites
resulted in an expression pattern without any nuclear targeting, and
the fusion protein equilibrated between the nuclear and cytoplasmic
compartments (Fig. 3F
) because it was of such low molecular
weight. This was the same expression pattern seen with EGFP alone
(compare Fig. 3, B and F
). Therefore, this region
of pRb2/p130 serves as a bipartite NLS where both the upstream and
downstream signals can independently dictate nuclear expression. Only
combined mutations in both the bipartite sites resulted in a complete
loss of exclusive nuclear expression. This is consistent with the
mutational data from the lymphoid cell lines, where both sites of the
bipartite NLS were disrupted by insertions or by point mutations.
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). On the contrary, the combined
mutations in both bipartite sites (pRb2/p130 NQ1&2-HA) almost
completely abolished the growth-suppressive activity of pRb2/p130
because the amount of cells in
G0-G1 was identical to that
of mock-transfected cells
(G0-G1 about 42%).
Addition of the c-myc major NLS to the COOH terminus of this
mutant (pRb2/p130 NQ1&2-HAN) by targeting its expression to the nucleus
restored its G0-G1 growth
arresting activity to that of the wild-type protein
(G0-G1 almost 60%). This
demonstrated that the reduced biological activity of pRb2/p130
conferred by these mutations was indeed conferred by their disruption
of the nuclear localization of pRb2/p130 and not by the induction of
gross conformational changes and/or the abrogation of binding to
critical protein targets. In addition, the growth-suppressive effects
of pRb2/p130 overexpression were not merely due to the toxicity of the
protein because point mutations in the protein effectively abrogated
this activity. Furthermore, pRb2/p130 must be in the nucleus to
regulate G1 progression. To rule out the
hypothesis that the growth-suppressive effects of pRb2/p130
overexpression could be because of mere toxicity of the protein, we
performed colony assays to study the long-term effects of mutant
pRb2/p130 overexpression. As shown in Table 3
, the mutant NLS-NQ1&2-HA that carries mutations within either the
upstream or downstream region of the bipartite NLS showed no
significant growth-suppressive activity with respect to the wild-type
pRb2/p130. Insertion of the c-myc major NLS into the COOH
terminus of mutant NLS-NQ1&2-HA to make the construct NLS-NQ1&2-HAN by
targeting its expression to the nucleus restored the growth arresting
activity to that of the wild-type protein in the colony assay,
confirming the results of the FACS analysis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The functional significance of the other mutations we found in osteosarcoma cell lines remains to be determined, even if they do not involve the NLS. Moreover, some mutations of the RB2/p130 gene were found in primary Burkitt’s lymphomas as well as in tumor-derived cell lines. These results demonstrate that the genetic alterations observed in tumor cell lines are unlikely to be purely a result of cell growth in culture.
While this work was in progress, the loss of pRb2/p130 in a small cell lung carcinoma cell line was reported (36) . This result supports the possible involvement of RB2/p130 gene alterations in lung cancer formation and/or progression (28) and strengthens the data on the genetic alterations of the retinoblastoma-related gene RB2/p130 that we found in different human tumors. In conclusion, these data clearly demonstrate genetic alterations in the RB2/p130 gene in tumor cell lines as well as in primary tumors, suggesting that alterations not only in the RB/p105 gene but in the RB2/p130 gene as well may contribute directly to the initiation and/or progression of the development of human lymphomas and leukemias. pRb2/p130 acts as negative regulator of cell cycle progression, blocking the cell in the G1 phase, and pRb2/p130 induction suppresses tumor growth (29) , as does pRb/p105, and its inactivation is fundamental for tumor progression. The members of the retinoblastoma protein family exhibit different growth-suppressive properties in specific cell lines and are not functionally redundant. Each member may play a unique and complementary role necessary to suppress neoplastic transformation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by 40% and 60% grants from the
Ministero dell’Università e Ricerca Scientifica, Instituti
Ortopedici Rizzoli "Ricerca Corrente" and "Foundations for
Selected Research Topics" (University of Bologna and Ferrara), and by
NIH Grants RO1 CA 60999-01A1 and PO1 NS 36466 and by Sbarro Institute
for Cancer Research and Molecular Medicine (to A. G.). P. P. C. is
the recipient of a fellowship from the "Associazione Leonardo di
Capua" (Naples, Italy). 
2 To whom requests for reprints should be
addressed, at Departments of Pathology, Anatomy, and Cell Biology,
Thomas Jefferson University, Philadelphia, PA 19107. Phone: (215)
503-0781; Fax: (215) 923-9626; E-mail: agiordan{at}lac.jci.tju.edu
3 The abbreviations used are: NLS, nuclear
localization signal; ATCC, American Type Culture Collection; SSCP,
single-strand conformational polymorphism; MDE, mutation detection
enhancement; TBE, Tris-borate-EDTA; HA, hemagglutinin; FACS,
fluorescence-activated cell-sorting; CMV, cytomegalovirus; EGFP,
enhanced green fluorescence protein.
Received 6/ 3/99. Accepted 11/11/99.
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REFERENCES |
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 D. S. Dimitrova and R. Berezney The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells J. Cell Sci., January 11, 2002; 115(21): 4037 - 4051. [Abstract] [Full Text] [PDF]
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 M. G. Paggi and A. Giordano Who Is the Boss in the Retinoblastoma Family? The Point of View of Rb2/p130, the Little Brother Cancer Res., June 1, 2001; 61(12): 4651 - 4654. [Abstract] [Full Text] [PDF]
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C. M. Howard, P. P. Claudio, Antonio De Luca, P. Stiegler, F. P. Jori, N. M. Safdar, M. Caputi, K. Khalili, and A. Giordano Inducible pRb2/p130 Expression and Growth-suppressive Mechanisms: Evidence of a pRb2/p130, p27Kip1, and Cyclin E Negative Feedback Regulatory Loop Cancer Res., May 1, 2000; 60(10): 2737 - 2744. [Abstract] [Full Text]
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P. P. Claudio, M. Caputi, and A. Giordano The RB2/p130 Gene: The Latest Weapon in the War against Lung Cancer? Clin. Cancer Res., March 1, 2000; 6(3): 754 - 764. [Abstract] [Full Text]
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 C. Cinti, L. Leoncini, A. Nyongo, F. Ferrari, S. Lazzi, C. Bellan, R. Vatti, A. Zamparelli, G. Cevenini, G. M. T osi, et al. Genetic Alterations of the Retinoblastoma-Related Gene RB2/p130 Identify Different Pathogenetic Mechanisms in and among Burkitt’s Lymphoma Subtypes Am. J. Pathol., March 1, 2000; 156(3): 751 - 760. [Abstract] [Full Text] [PDF]
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