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From the Departments of Pathology*
and
Biochemistry and Molecular Biology,
University
of Southern California School of Medicine, Los Angeles, California
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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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Related to the observation of L-plastin overexpression in prostate cancer, another report describes that a new member of the thymosin ß family (thymosin ß15) of G-actin binding molecules is up-regulated in highly motile and metastatic Dunning rat prostatic cancer cell lines.10 Transfection of antisense thymosin ß15 constructs into rat prostate carcinoma cells reduces cell motility. Furthermore, thymosin ß15 levels appear to be elevated in human prostate cancer and correlate positively with the Gleason tumor grade. As cell motility is likely to be linked to coordinated diassembly and reformation of the cortical actin network, it has been proposed that binding of thymosin to actin may enhance the depolymerization phase of this process.10
We hypothesized that the uniform overexpression of L-plastin in prostate cancer is likely to have a role in prostate cancer progression by directly or indirectly influencing growth, invasion, and metastasis of the tumors. As a first step in this line of investigation, we report an evaluation of the functional consequence of L-plastin overexpression in prostate cancer cell lines by suppressing the L-plastin expression. The results obtained indicate that in vitro characteristics examined, namely, proliferation efficiency and Matrigel invasion, are inhibited by antisense L-plastin sequence expression. Furthermore, we present data to indicate that L-plastin down-regulation manifests a negative influence on in vivo invasion of prostate tumor cells in an experimental animal model. Thus, a clinical potential exists that this antisense sequence may suppress progression of human prostate tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prostate carcinoma cell line PC-3 and its metastatic variant subline PC-3M were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), streptomycin-penicillin, glutamine, 1X non-essential amino acid solution, and 2X essential amino acid solution (Life Technologies, Grand Island, NY) as described.11 After transfection with antisense L-plastin constructs or infection with an antisense virus, the cells were selected and maintained with 400 µg/ml G418 in DMEM culture medium.
Construction of Retrovirus Expression Vectors
A 163-bp fragment containing 137 bp of the 5'-untranslated region
and 26 bp of the adjacent 5'-translated region of human L-plastin was
produced by reverse transcriptase polymerase chain reaction (RT-PCR)
with the sense primer C07 (5'-GAATTCACTTCCTGCCTTGTGACCAC-3') and the
antisense primer C10 (5'-GAATTCTCATCGGACACTGATCCTC-3'). Both primers
were designed to contain an EcoRI restriction site at 5'
end. The PCR product was then digested with EcoRI and cloned
into the EcoRI site of the pWZLneo retroviral
vector.12,13
A 1713-bp fragment composed of the 3'-coding
region sequence of human L-plastin was also produced by RT-PCR as
described5
and cloned into TA cloning vector (Invitrogen,
Carlsbad, CA). The insert was then cut out of the vector by
EcoRI and cloned into the EcoRI site of the
pWZLneo vector. The antisense constructs for both fragments, the
3'-coding region (AS1) and the 5'-untranslated region (AS2), were
confirmed by sequence analysis. A scheme of the pWZLneo retroviral
vector and both antisense constructs are shown in Figure 1
.
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Each of the antisense constructs and the vector-alone plasmid DNA were transfected into PC-3 and PC-3M cells by Lipofectamine (Life Technologies) following the manufacturer's protocol. After 48 hours, the cells were split 1:10 and selected in DMEM containing 600 µg/ml Geneticin for a period of 3 weeks. As the selection of transfected cells was based on the expression of the downstream Neo resistance gene product from the same bicistronic proviral DNA carrying the antisense sequence, the use of the WZLneo vector allowed virtually all selected resistant cells to express the antisense RNA. At least 60 colonies were pooled from each transfection and expanded to generate the AS1, AS2, or vector-only stable cell lines used in the in vitro growth and invasion assays.
Isolation and Utilization of Retrovirus Containing the AS2 Sequence
The AS2 or the vector-only DNA was transfected into PA317 amphotropic retrovirus packaging cell line by Lipofectamine, and after 48 hours the cells were split into 1:10 and cultured in DMEM containing 600 µg/ml Geneticin. The surviving clones were trypsinized and expanded individually. The viral titer of the PA317 clones was determined by transfer of the G418 resistance to NIH3T3 cells using serial dilutions of viral supernatant. Clones of cells producing viral titers between 3 x 105 and 5 x 105 infectious units per milliliter were selected for virus collection and for use in the experiments described.
The harvested virus supernatants for AS2 or vector-alone constructs were used to infect PC-3 and PC-3M cells at a multiplicity of infection of approximately 10 to 25 in the presence of 8 µg/ml polybrene, and the infection procedure was repeated three times for the same culture. The infected cells were selected in the medium containing 600 µg/ml Geneticin, and individual drug-resistant clones were isolated and expanded for further study. For each construct and each cell type, 15 clones were picked for analysis of L-plastin expression.
Western Blot Analysis
Transfected or infected prostate tumor cells grown in one T75 culture flask were lysed in 250 µl of 2X Laemmli buffer. After boiling for 5 minutes, 10 µl of each lysate was loaded onto a 10% SDS-polyacrylamide gel to resolve proteins. Separated proteins were then transferred to nitrocellulose membranes. The expression of the L-plastin protein was determined using a rabbit anti-L-plastin antibody as previously described.5 The membranes were stripped and reprobed with ß-actin (Sigma Chemical Co., St. Louis, MO) to assess the protein loading for each lane. The intensities of the bands were determined using NIH Image software. The ratio of the intensity of each L-plastin band from different clones over the intensity of the corresponding ß-actin band was calculated and compared for an increase or decrease in L-plastin expression.
Cell Proliferation Assay
Pooled clones of cells transfected with vector-alone or with antisense constructs were seeded at a density of 105 cells/60-mm dish in triplicate. The growth rates were monitored over a period of 7 days. Cells were trypsinized every day from the respective plates and counted using a hemocytometer. Each experiment was repeated twice.
Tumor Cell Migration and Invasion Assay
To investigate the effects of decreased expression of L-plastin on the ability of PC-3 and PC-3M cells to migrate through a filter or to invade a biological barrier, we prepared 24-well invasion chambers using Matrigel basement membrane matrix (Becton Dickinson Labware, Bedford, MA) following the manufacturer's suggested procedure. The invasion chamber consists of a 24-well plate with inserts containing an 8-µm pore-size membrane with a thin layer of Matrigel basement membrane matrix, which contains laminin, collagen type IV, heparin sulfate proteoglycan, entactin, and growth factors, including transforming growth factor (TGF)-ß and fibroblast growth factor (FGF)-2. Cells were suspended in DMEM containing 0.1% bovine serum albumin and added to the upper chamber at 1 x 105 cells/insert. A conditioned medium obtained by incubating NIH 3T3 cells for 24 hours in serum-free DMEM in the presence of 50 µg/ml ascorbic acid was placed in the lower compartment of invasion chambers as chemoattractants. After 24 hours of culture, the upper surface of the inserts were wiped with cotton swabs, and the inserts were stained with hematoxylin and eosin (H&E). Each experiment was performed twice with each sample in triplicate. The cells that migrated through the Matrigel and the filter pores to the lower surface were counted under a light microscope with five random high-power fields per insert.
In Vivo Diaphragm Invasion Assay
The diaphragm invasion assay was performed as described previously.14 Briefly, the cell lines to be tested were grown in culture, trypsinized, washed twice in phosphate-buffered saline, and counted by hemocytometer. Aliquots of 1 x 106 cells were injected intraperitoneally into nu/nu mice, and tumors were allowed to develop for 21 days. In a single experiment, groups of five animals were injected with each of the following cell clones: PC-3 derivatives (vector virus infected V4 and V9 clones and AS2 virus infected 5.4, 8.5, and 10.4 clones) and PC-3M derivatives (vector virus infected VM8 and VM10 clones and AS2 virus infected 11.2, 11.3, and 11.6 clones). At 21 days after inoculation, all animals except two that died before the termination point were sacrificed, the diaphragm from each animal was removed, and the tissue was formalin fixed. Before paraffin embedding, each diaphragm was cut into four sections and arranged in the block such that each paraffin section would contain four different areas of a single diaphragm. Each diaphragm was sectioned three times at different levels of the paraffin block, thus yielding 12 random sections of each diaphragm. The cross sections of the diaphragm were examined for tumor colonies and scored on the basis of basement membrane penetration. A minimum of 12 cross sections from different areas of each diaphragm were examined, and evidence of invasion in any visible tumor resulted in a positive score for invasion. The use of a single event of invasion rather than a percentage of invasive tumors/diaphragm was used to decrease the chances of overestimating the power of the assay. Statistical significance between vector-only and AS2 clones was determined by Fisher's exact test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Levels of expression of L-plastin in antisense transfected or
antisense retrovirus infected PC-3 and PC-3M cells were examined by
Western blot (Figure 2)
. Figure 2A
shows
the results for pooled transfected cells. A higher level of L-plastin
expression was seen in the metastatic variant PC-3M cells as compared
with the parental PC-3 cells. Although expression of vector alone did
not appear to change the levels of L-plastin expression in either PC-3
or PC-3M cells, there was a significantly lower expression in cases
where the cells were transfected with the antisense constructs.
Although both of the antisense constructs were effective, the 163-bp
5'-untranslated region (AS2) showed a stronger effect on the reduction
of L-plastin expression than the 1713-bp coding region (AS1). Figure 2B
shows the results from individual AS2 virus infected clones of PC-3 and
PC-3M cells. The intensities of the bands recorded on gel pictures were
measured and compared by NIH Image software for these clones. The
expression of L-plastin protein was reduced by approximately 4.5- to
10-fold in antisense L-plastin infected PC-3M and by 3.2- to 10-fold in
antisense L-plastin PC-3 cells relative to the corresponding
vector-only virus infected clones.
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Antisense L-plastin transfected pools of PC-3 and PC-3M cells were
compared for their proliferative abilities with those of vector
transfected or untransfected PC-3 and PC-3M cells. At low cell
densities, the expression of either antisense L-plastin RNA could
reduce the proliferation rates of the transfected cells by
approximately 30% to 40% as compared with the vector transfected or
untransfected cells as shown in Figure 3
.
The reduction of the proliferation rate appeared to be slightly greater
in antisense transfected PC-3 cells relative to similarly treated PC-3M
cells. There was, however, no significant difference in growth rate
with respect to AS1 or AS2 expression in either of the cell lines. The
down-regulation of L-plastin expression did not induce any obvious
morphological changes to PC-3 and PC-3M cells.
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Invasion chambers were used in this study to investigate the
influence of antisense L-plastin expression on the abilities of PC-3
and PC-3M cells to migrate and invade in an in vitro system.
After 24 hours of culture, cells that invaded through basement membrane
Matrigel and migrated into the other side of the pored filters were
checked under a microscope, and the total cell number for each filter
was counted. The pooled cells with antisense L-plastin showed drastic
reduction of cell number as compared with pooled vector transfected
cells (Figure 4)
. Both AS1 and AS2
constructs produced a significant reduction in invasion and migration
properties, although AS2 caused more reduction (87%) than AS1 (68%)
in PC-3 cells. The AS2 expression also caused a reduction in invasion
(89%) in PC-3M cells relative to AS1 expression (81%). No significant
morphological changes in either PC-3 or PC-3M cells were observed.
Results of comparative invasiveness for AS2 retrovirus and vector virus
infected clones of PC-3 and PC-3M cells are presented in Figure 5
. Suppression of invasion for either
PC-3 (P < 0.05) and PC-3M
(P < 0.01) was determined to be significant by
the Student t-test analysis.
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To determine whether the property of reduced in vitro
invasion by antisense L-plastin-expressing clones is also maintained
in vivo, we used a previously described in vivo
model of invasion in nude mice.14
Three different clones
of each of the PC-3 and PC-3M cell lines infected with AS2 retrovirus
were analyzed along with two clones each of vector-only controls.
Groups of four animals were injected intraperitoneally with equal
amounts (106) of cells of each of the clones. After 21 days
of incubation, diaphragms were examined for tumor colonies. As
indicated in Figure 3
, there was a growth difference between vector and
AS cell lines, and this difference was maintained in vivo.
The tumors from vector-only cells were larger than AS2 tumors, but the
number of tumor colonies/diaphragm did not appear to be different. The
number of diaphragms analyzed in the various groups were as follows:
PC-3 vector, 8; PC-3 AS2, 11; PC-3M vector, 7; PC-3M AS2, 13. This
analysis revealed that both PC-3 and PC-3M cells were indeed invasive
in the assay system used. Figure 6
illustrates this issue (Figure 6, A, B, and E)
with vector virus
infected PC-3 and PC-3M cells. On infection with AS2 virus the tumors
were determined to be less invasive with the majority of diaphragms
having no evidence of invasion as illustrated in Figure 6, C–E
.
Approximately 80% and 70% reduction in invasion was noted in AS2
virus infected PC-3 and PC-3M cells, respectively, compared with the
corresponding vector-only controls. These differences were determined
to be statistically significant (P = 0.006 for
PC-3 and P = 0.005 for PC-3M).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Second, it is exciting to find that suppression of L-plastin expression in prostate cancer cells can drastically decrease in vitro invasion and motility of these cells. These in vitro results are strengthened by the observation that antisense-mediated reduction of L-plastin expression in PC-3 or PC-3M results in significant suppression of in vivo invasion as assayed by an immunodeficient mouse model of tumor cell invasion. The reduction in diaphragm invasion was approximately fivefold in PC-3 cells and threefold in PC-3M cells. This is an important observation, but the mechanisms by which L-plastin down-regulation inhibits invasion remain unknown. It is reported that cadherin requires anchoring to the actin-based cytoskeleton through catenin to mediate cell-cell adhesion.19,20 Furthermore, focal adhesion points are, in fact, adhesive cell membrane structures in which transmembrane receptors such as integrins are clustered to connect the intracellular actin cytoskeleton to the extracellular matrix.15,16 Conceivably, because of its binding capability to cytoskeleton structure, L-plastin, when overexpressed, alters the cytoskeleton structure.9 This in turn leads to disruption of cell-cell adhesion or cell-extracellular matrix interactions. Conversely, underproduction of L-plastin may restore strong cell-cell and cell-matrix interactions, resulting in reduction of invasive and migratory properties of the affected cells. Although it is speculated that reduction in L-plastin levels affects the actin cytoskeleton and perhaps secondarily adhesion plaques, we do not have any direct evidence in support of those ideas at this time. Obviously, further work will be necessary for elucidating the underlying mechanisms.
Finally, our previous evidence of uniform overexpression of L-plastin in prostatic malignant epithelium,5 combined with the present evidence of inhibition of in vitro cell growth, motility, and invasion and in vivo tumor cell invasion by reduction of L-plastin expression, presents a case of strong clinical relevance for this line of work. We would like to project that by using vectors with high gene transfer efficiency coupled with strategies of prostate tissue-specific gene expression, it might be possible in the near future to inhibit L-plastin-related motility and invasion properties of the prostate cancer cells, thereby suppressing prostate cancer progression. A perceived advantage of the approach, in a clinical sense, would be to create a window of opportunity to treat prostate cancer by conventional protocols while tumor cell migration is stalled.
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Acknowledgements |
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Footnotes |
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Supported by U.S. Public Health Service grant CA 59705 from the National Cancer Institute and, in part, by postdoctoral fellowships from the NCI training grant T32- CA09320 (to J. Zheng), NIAID training grant T32-AI07078 (to W.C. Powell), and a grant from the T.J. Martell Foundation.
J. Zheng and N. Rudra-Ganguly contributed equally to this work.
Accepted for publication March 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev 1994, 8:1654-16632-Adrenoceptor stimulation promotes actin-polymerization and focal adhesion in 3T3F442A and BFC-1ß preadipocytes. Endocrinology 1996, 137:5220-5229[Abstract]
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