1. Human Telomerase Reverse Transcriptase (hTERT) Transfection Reduces Apoptosis in Human
Corpus Cavernosum Smooth Muscle Cells Apoptosis and Slows Ddown the Cellular Aging
Abstract
Aims: To explore effects of hTERT gene transfection on biological behaviors of human corporal smooth
muscle cells.
Methods: Human corporal smooth muscle cells were grown in primary cultured; . aA fluorescent
eukaryotic expression vector, named hTERT-IRES2-EGFP, of carrying the hTERT gene was constructed
and; for the first time, the sense recombinant plasmid hTERT-IRES2-EGFP was transfected into human
corporal smooth muscle cells with using Lipofectin reagent.; the correctness of transfection was
identified, as well astThe telomerase activity, mitotic index, cell apoptosis, cell growth curves of
transfected smooth muscle cells were determined;, and whether the potential formation of malignant
phenotypes occurred in these transfected cells were determinedwas investigated.
Results: We successfully constructed fluorescent a eukaryotic expression vectors of human sensecarrying
hTERT and EGFP genes and correctly transfected human corporal smooth muscle cells. Telomerase
activity, mitotic index, and cell growth curves of the hTERT-transfected cells were significantly higher
than those of non-transfected cells and of cells transfected with vacant the empty EGFP vector EGFP-
transfected cells, , whileereas cell apoptosis rates of the former were lowest inr hTERT-transfected
cellsthan the latter. No changes in cell morphology, chromosome number, and The hTERT-transfected
cells still had 23 pairs of chromosomes and all were normal diploid cells. No tumorigenicity in nude mice
wereas foundobserved between hTERT-transfected cells and control cells.
Conclusion: In this study, for the first time, the sense hTERT gene was transfected into human corporal
smooth muscle cells, which helped increased telomerase activity in cells, reduced cell apoptosis and
slowed down cell aging. No malignant phenotypes were found in the hTERT-transfected smooth muscle
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2. cells.
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3. Introduction
Erectile dysfunction (ED) is one of the most common diseases in Male Urology that greatly affects
the quality of life in aging men. Due to accelerated As aging of global population accelerates, the number
of patients with ED have beenis increasing year byeach year, which greatly affects quality of life of aging
males. Senescence has been generally recognized as a high-risk factor for ED [1]
. Relaxation of corpus
cavernosum smooth muscle is the key to penile erection. Although one PED5I, a phosphodiesterase V
inhibitor (PED5I), has been used toyielded good results in the treatment of ED, achieving good results,
but its has poor efficacy in patients with for organic ED was poor [2]
. At present, the mechanism of organic
changes in ED is not yet known. Our research and related foreign literature and others have found a
decrease inthat in the process of aging and the occurrence of organic ED, smooth muscle cells (SMCs) in
penile tissues decreased, an increase in collagen fibers increased, and a decrease in erectile tissue
compliance in the process of aging and with the occurrence of organic EDdecreased. There is currently no
ideal way to reverse the above changes yet [3]
. Current evidence Most of viewpoints supports the
viewpoint that a the decrease inof SMCs in penile tissues of patients with ED is due to the transformation
from SMCs to fibroblasts. However, injection of antisense TGFβ to inhibit smooth muscle fibrosis has
poor effects.
An increase or a decrease in the number of cells depends on the speed rate of cellular aging. In
recent years, theWith the progress in genetics, of aging have made new progress, in which one very
important thing is the discovery of telomeres and telomerase have been implicated in the regulation of
aging. Iwana et al. x have confirmed that telomere shortening acts as the molecular clock that triggers
senescence. Telomerase can catalyze and replicate telomeres, and thereby extend cellular life-span.
Among the three known subunits of comprising human telomerase, only the human telomerase protein
catalytic subunit (hTERT), an important rate-limiting component of human telomerase, is consistent
associated with telomerase activity [4]
. Due to the lack of telomerase activity in human penile tissues,
3

4. telomeres in SMCs gradually shorten along with increasing cycles of mitosis. The lLife-span of SMCs
with a certain level of telomerase activity may, therefore, be extended. However, Nno related research has
currently been reported till date.
In the present This study, we targeteds primary cultured human corporal smooth muscle cells. The
fluorescentAn eukaryotic expression vector, named hTERT-IRES2-EGFP, carrying theof hTERT gene
and the gene encoding enhanced green fluorescent protein (EGFP) was constructed and identified; for the
first time, the sense recombinant plasmid hTERT-IRES2-EGFP was transfected into human corporal
smooth muscle cells.; We then evaluated thethe correctness of efficiency of transfection was identified
and determined, as well as telomerase activity, mitotic index, and cell apoptosis, as well as measured cell
growth curves of transfected smooth muscle cells. In addition, we examined, and whether malignant
phenotypes occurred in these transfected cells were determined.
Materials and Methods
Primary culture and identification of human corporal smooth muscle cells
The human penile corpus cavernosum was obtained during hypospadias surgery. The cavernosal
tissue was cut into small pieces (~approx. 1-3 mm each), and then placed for 10 min in PBS solution
containing 100 U/ml penicillin and 100 μg/ml streptomycin for 10 minutes. The tissue pieces were
thereafter placed at 0.5 cm intervals into 25 ml sterile culture bottles coated with 20% fetal calf serum
(FCS) and fixed. Then, 1.5 ml prepared Dulbecco's Modified Eagle's Medium (DMEM) containing 20%
FCS, 100 U/ml penicillin and 100 μug/ml streptomycin was then added to each bottle, and thereafter
theythe culture bottles were placed incubated at 37o
C in aunder 5% CO2 incubator at 37 ℃. After 3-5
days of incubation, we observed the tissue pieces under an inverted microscope. When a few cells were
found to be non-adherentswim out of the plant, we added more medium, as well as recorded and observed
the cell morphology and growth. When the cells reached 90% confluence, they were mixed with 0.5%
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5. trypsin and 0.04% EDTA (1:1), and were then passaged after the digestion.
Construction and identification of sense fluorescent eukaryotic expression vector hTERT-IRES2-
EGFP
The full-length sequence of hTERT cDNA was located between two EcoRIⅠ restriction sites inof
plasmid pGRN145 (a kind gift from Geron Inc., USA). After digestioned from plasmid pGRN145 and
purifiedpurification, the fragment containing hTERT was cloned into the EcoRI Ⅰsites of the fluorescent
eukaryotic expression vector pIRES2-EGFP. Plasmids with the desired oOrientation of the insert
wereconnection was determined by the digestion with Not I restriction endonuclease followed by agarose
gel analysis.
The hTERT gene Ttransfection of hTERT gene into SMCs
The Lipofectin kit (Gibco) was used for this transfection. One day before transfection, the cells were
passaged to 24 cm2
flasks to make theseso that cells reached around 50% confluence the next day.
Lipofection complexes were prepared as follows: 100 μl of medium containing 13 μl of Lipofectin was
placed at room temperature for 45 minutes, and was then mixed with 100 μl of medium containing 2.2 μg
of plasmid DNA. The mixture was placed incubated at room temperature for 15 minutes. The cells to be
transfected were then added to this mixture, and the transfection mixturereafter were putwas incubated at
37o
C under in a 5% CO2 incubator at 37 ℃. Twenty-four hours after the incubationlater, we changed the
medium was changed and then maintained the culture was maintained for another 48 hours. Then, G418
was added and selection continued to screen until colonies formed.
Detection of EGFP expression in cells by confocal laser scanning microscopy
Normal SMCs transfected with the vector hTERT-IRES2-EGFP , SMC-hTERT and SMC-EGFP
were respectivelywere observed under a Leica TCL-NT confocal laser scanning microscope to detect
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6. examine whether fluorescence fluorescent protein expression and morphological changes occurred in
cells. As controls, untransfected SMCs and SMCs transfected with xx and yy were used.
Detection of integration of exogenous NeoR gene in cells by PCR
This The detection was conducted using the xx kit according to the kit instructions provided by the
supplier and th. The following upstream of NeoR gene-specific primers: was 5'-
CAAGATGGAATTGCACGCAGG-3' (forward) and downstream 5'-CCGCTCAGAAAGAACTCGTC-3'
(reverse).
Detection of expression of hTERT mRNA levels expression by RT-PCR
This The detection was carried out using the xx kit according to the kit instructions provided and.
The upstream ofthe following hTERT gene-specific primers was : 5'CGGAAGAGTGTCTGGAGCAA3'
(forward) and downstream 5'GGATGAAGCGGAGTCTGGA3' (reverse).
Detection of exogenous hTERT protein expression in transfected SMCs by Western blot
When normal untransfected or transfected SMCs, pIRES2-hTERT-SMC and EGFP-hTERT-SMC
reached 90% confluence, total cellular protein was isolated using M-PERTM
(Pierce), a mammalian protein
extraction reagent (Pierce), was respectively administered at these cells to collect total cellular protein.
After protein quantification by using BCA protein assaymethod, samples were separated by SDS-PAGE,
electrotransferred electrically onto a nylon membrane,s and incubated with anti-hTERT immune antibody
responses were performed. EventuallySubsequently, enhanced chemiluminescence (ECL) was used to
visualize protein bands.
Quantitative detection of telomerase activity by TRAP-ELISA
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7. This detection was performed according to the instructions.
Detection of changes in the number of SMC proliferation by mitotic index
The mitotic index of SMCs was calculated by staining cells and counting the number of cells in
mitosis, respectively, at different time points.
Detection of absorbance changes in SMC proliferation by MTT assrray
This detection was conducted in a conventional mannerusing standard procedures. One multi-well
plate was colored each day for a total of 6six days, then 20 υµl of 5 mg/ml MTT was added to each well,
and then plates were incubated at 37 ℃ for 4four hours at 37o
C. After the incubation, 150 µul of dimethyl
sulfoxide (DMSO) was added to each well and then plates were shaken for 10 minutes to make crystals
fully dissolve crystalsd. The absorbance of each well Plates were readwas then measured at 490 nm
usingwith an enzyme-linked immunosorbent assay (ELISA plate) reader (490nm) to measure the
absorbance of each well. Cells growth curves were determined by plotted with time as x-axis anding
optical density (OD) values as y-axisa function of time.
Detection of SMC apoptosis using TUNEL staining methodassay
This detection was carried out according to the kit instructions.
Karyotype aAnalysis of chromosome karyotype
Colchicine was added A bottle of to SMC-hTERT cells grownup to the logarithmic phase was added
by colchicine., and Followingsubsequently incubated incubation for 2 hours at 37o
C ℃ for 2 hours, and
then the cells were collected as well asand treated with hypotonic solution. ThereafterSubsequently, the
cells were prefixed with ice-cold methanol / acetic acid (3:1) solution for 5 minutes, deposited by
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8. centrifuged centrifugationdeposit, and fixed again. FinallyNext, they were made into a single cell
suspension. The single cell suspension, was dropped onto a pre-cooled glass slide, stained with Giemsa
stain for 10 -15 min,utes and then observed under the an optical microscope.
Experiment of Ttumorigenicity in nude mice
Six nude mice of either sex, raised under specific pathogen-free (SPF) conditions, were randomly
divided into 3 three groups (2 two mice per group), raised under specific pathogen free (SPF) conditions.
All groups of nudeEach mice mouse were was inoculated subcutaneously with the liver cancer cell line
HepG2 into the left backs hind legas a control., The different groups then receivedwhereas with the same
amount of untransfected SMCs or SMCs transfected with -hTERT or , SMC-EGFP and SMC into the
right hind legbacks. Subsequently,The tumor formation was observed and meanwhile tumor size was
measured every 7 seven days for successive 8 eight consecutive weeks.
Statistical analysis
Experimental data were analyzed using SPSS 13.0 software and represented as mean ± SDsd (x ±
s). When the means of several groups were compared, single-factor analysis of variance was used for
homogeneity of variance. The q-test was used for the comparison of two means. The A non-parametric
test was performed for heterogeneity of variance. A P value less than <0.05 was considered statistically
significant. , while a value A P<less than 0 .01 was considered highly significant.
Results
Primary culture and identification results of human corporal smooth muscle cells
On the 4th
day of primary culture, a few spindle cells grew out of the tissue edge and; on on the 5th
to 7th
days, an increase in the number of the cells increased in the number and beganand the to appearance
of a directional arrangement was observed. After around 2 two weeks of culture, a dense monolayer of
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9. cells formed in the flask and cells were passaged. After the passage, some cells were spindle-shaped and
showed directional arrangement (Fig. 1). The cultured cells were proven to be SMCs by
immunohistochemical staining with a monoclonal antibody against α-actin. Both Sspindle-shaped SMCs
with a pale yellow cytoplasm and actin stained dark yellow staining of actin in cells were both observed
under the microscope (Fig. 2).
Construction and identification results of sense and antisense hTERT eukaryotic expression vector
Digestion Identification of Plasmid 1pGRN145 and IRES2-EGFP
The plasmid pGRN145 containing 3.5-kb hTERT cDNA between the two EcoRIⅠ restriction sites of
pBBS212 was is 14-kb long. The plasmid pGRN145 should form two fragments (11.5 and 3.5 kb) after
digestion with EcoRI Ⅰ. The vector IRES2-EGFP produced a 5.3-kb linear fragment after digested
digestion withby EcoRI, Ⅰand formed two fragments (4.0 and 1.3 kb) after double digestion with EcoRIⅠ
and NotIⅠ. The experimental results were completely consistent with theoretical values. (sSee Fig. 3 and
Fig. 4).
Digestion identification of the recombinant plasmid hTERT-IRES2-EGFP
In this experiment, Bbased on cloning into an EcoRIⅠ restriction site, the inserted fragment carrying
hTERT cDNA can be forward or reversely connectedoriented in two directions to the fluorescentin
the eukaryotic expression vector., so Hence, the Not I restriction endonuclease digestion was further used
for to identification identify theof the connection orientation of the insert in different clones. The sense
plasmid was found with two bands of 1.4 and 7.4 kb, whereas the antisense plasmid has two bands of
with 4.8 and 4.0 kb, respectively (. The two bands were consistent with theoretical calculations. (See Fig.
5).
The Transfection of sense recombinant plasmid hTERT-IRES2-EGFP transfection into human
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10. corporal smooth muscle cells
By using the cationic liposome Lipofectin, the sense recombinant plasmid and vacant empty vector
were transfected into cultured SMCs; the corresponding cells were, named as SMC-hTERT and SMC-
EGFP, respectively.
Identification of Ttransfection results
● Observations results underby fluorescence microscopye
Forty-eight hours after transfection, the positive expression of EGFP was observed under
inverted fluorescence microscope. The entire cell appeared green, and nucleus-based (Fig. 6). No change
in and its cell morphology was consistent with that ofas compared to non-transfected SMCs was
observed. The entire cell appeared green, and nucleus-based. (See Fig. 6)
● Reporter gene assay
Our results showed that both SMC-hTERT and SMC-EGFP were observed to respectively
produced a specific PCR amplicon band of 790 bp, while SMC had no amplified bands. This suggested
that the sense plasmid hTERT-IRES2-EGFP and vacant vector had already beenwere successfully
transferred into SMCs (. (See Fig.7).
● Identification of the hTERT transcriptional level expression
The expression of the hTERT gene in transfected SMCstranscription products was detected by RT-
PCR assay. Our results showed that SMC-hTERT as well as SMC and SMC-EGFP all had
expressionexpressed this genes, but that the level of hTERT mRNAexpression was significantly higher in
SMC-hTERT was significantly higher than that in SMC or in SMC-EGFP. This result indicated that
primary cultured SMC endogenously expressed had hTERT at a low level, and that transcription but
exogenous transfected hTERT gene transfection could significantly increase its the transcription of this
gene in SMCs. (See Fig. 8).
● Identification of protein level
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11. The level of hTERT protein expression in hTERT-transfected cells was significantly higher in
hTERT-transfected cells than that in non-transfected cells. This result suggested suggests that the
increased level of hTERT mRNA in hTERT-transfected cells exogenous hTERT gene can becorrelates
with the level of translated protein in human corporal smooth muscle cells (. (See Fig. 9).
Quantitative detection measurement of telomerase activity
Telomerase activity in hTERT-transfected cells was significantly higher than that in non-transfected
cells or in vacant vector EGFP-transfected cells transfected with the empty x vector (0.270 ± 0.40 vs.
0.120 ± 0.011, 0.092 ± 0.026, P < 0.01). This data indicated that exogenous hTERT
transfection into SMCs can significantly increase the telomerase activity.
Detection of changes in the number of SMC proliferation by mitotic index
After the hematoxylin staining, lamellae-crawling SMCs were counted for cells in mitosis under the
light microscope, and the percentage of the cells in mitosis was calculated. (sSee Table 1 and Fig. 10).
Detection of changes in absorbance of SMC proliferation by MTT assrray
After 7 days of culture, the OD value of SMC-hTERT was significantly higher than that of SMC-
EGFP or that of SMC, whereas no significant difference in OD value was found between SMC-EGFP and
SMC in the OD value. (sSee Table 2 and Fig. 11).
Detection results of SMC apoptosis by TUNEL staining
After the logarithmic growth phase of a cell (5 five days), the occurrence of apoptosis was
determined in SMC-hTERT, SMC-EGFP, and SMC could be detected occurrence of apoptosis., T and the
apoptosis in SMC-hTERT was significantly less in SMC-hTERT than that in either SMC-EGFP or
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12. SMC (2.80 ± 0.04 vs. 6.33 ± 0.01, 6.53 ± 0.17, P <0.01).
KAnalysis of chromosome karyotype analysis
After long-term passages in vitro, each SMC-hTERT had 46 chromosomes, the same number as a
normal somatic cell has.
TExperiment of tumorigenicity in nude mice
Eight weeks after inoculatjectioned with 4×106
HepG2 cellseach, the left sides of all nude mice
injected with HepG2 cells formed tumors (i.e., 100%, with the tumor formation rate of 100%;). By
contrast, whereas their right subcutaneous tissues that were injected with either SMC, SMC-EGFP and or
SMC-hTERT had did not form zero any tumors formation rate.
Discussion
HThe human telomerase is at least composed of human telomerase RNA (hTR), telomerase-
associated protein 1 (TP1), and the telomerase protein catalytic subunit, human telomerase reverse
transcriptase hTRT (or also termed hTERT or, hEST2). The component hTR in telomerase may mainly
act as a template. The subunit TP1 also widely exists in all tissues, and it is, therefore, not unlikely to
functions as an important regulator of telomerase. In 1997, Meyerson and co-workers [5]
and Nakamura [6]
et al.[6]
independently in different laboratories cloned respectively another protein subunit of telomerase--
the telomerase protein catalytic subunit hTERT at the same time. The hTERT gene iwas located as a
single copy at 5p15. 33, and its full-length cDNA iwas 4027 bp long in size, with a full-length open
reading frame of 3399 bp, encoding 1132 amino acids. The hTERT containing contains 7seven small
functional domains and is a memberone of the reverse transcriptase family members with reverse
transcriptase activity. Some Several studies have shown that different from the genes hTR and TP1, the
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13. hTERT gene is only expressed in tumor cells, immortal cells, and germ cells/stem cells, but not
in telomerase-negative normal tissues, which has a veryin agreement good consistency withwith data
regarding telomerase activity [7-8]
. The hTERT and hTR were reconstructed in vitro, and the artificially
constructed telomerase also had the function of telomere repeat synthesis. Inducers of tTumor cell
differentiation inducer can decrease telomerase activity by downregulating hTERT gene expression, while
the expression of either hTR or TP1 remained unchanged. The above studies suggest that the hTERT gene
is a major rate-limiting component of telomerase.
Normal somatic cells do not express hTERT, and are telomerase negative. The telomeres in those
cells gradually shorten along with increasing cycles of mitosis, thus leading to cell aging and its finalcell
death. Research has shown that the introduction or activation of the hTERT gene can activate telomerase
in somatic cells, and thereby maintaining telomere length or slow downdelaying loss of telomere losss [9-
11]
. Bodnar et al. x used vectors encoding human telomerase catalytic subunit to transfect two types of
normal human somatic cells without telomerase activity, retinal pigment epithelial cells and foreskin
fibroblasts. It wasThey found that the transfected cells restored telomere activity, that the length of the
sequence repeats TTAGGG repeat tract at chromosome ends rapidly increased, and that DNA cells with
artificially elongated telomeres had significant changes in growth potential. In their experiments, normal
somatic cells as a control started aging along with telomere shortening after limited cell divisions,
whereas telomerase-expressing clones lost normal senescence and continued dividing vigorously. The
life-span of many clones reached was at least as 20 times longer as than that of normal cells. More
importantly, the transfected cells did not have malignant phenotypes of tumor cells, owned possessed the
exactly the same chromosome karyotype as that of normal cells, had no tumorigenicity in nude mice, and
possessed a variety of differentiation functions comparable to those of normal cells had[12]
.
The plasmid pIRES2-EGFP also contains IRES and EGFP in addition to the necessary components
for a eukaryotic expression vector. The The EGFP gene, a commonly used reporter gene, is a green
fluorescent encodes a protein composed of 238 amino acids that exhibits a bright green fluorescence after
13

14. excitation by UV. It is commonly used as a fluorescent reporter This can make researchersto rapidly
assess the transfection efficiency by monitoringdetecting EGFP expression in cells. The component
IRES can start initiate the expression of respectively two or more proteins expressions of
the polycistronic transcription unit in eukaryotic cells at the same time, and the
proteins function independently of each other. Therefore, in this study, we chose pIRES2-EGFP as a
vector for, the inserted insertion of the hTERT cDNA fragments into multiple cloning sites of the plasmid
pIRES2-EGFP by genetic recombination, and successfully constructed the recombinant eukaryotic
expression vector pIRES2-EGFP-hTERT. In this study, we successfully primary cultured human corporal
smooth muscle cells using the tissue block method, and, for the first time, transfected the pIRES2-EGFP-
hTERT was transfected hTERT gene into human corporal smooth muscle cells using cationic liposome
methods. Meanwhile,Transfection and target protein expression were verified by detecting more a strong
fluorescent signalsubstances in the transfected cells were observed underusing a fluorescence microscope,
and by Western blot and telomerase activity assaysconfirming that the target gene products were
expressed after transfection. Using RT-PCR and Western blot assaysanalyses furthermore showed, we
found that non-transfected SMC and SMC-EGFP did not haved a very low level of hTERT
mRNA expression, but while SMC-hTERT steadily had the expressioned hTERT. This result
demonstrated that the exogenous hTERT gene could be steadily transcribed and expressed in SMCs.
Reconstruction of telomerase activity in normal somatic cells can delay cell aging; however,, but its
overexpression is an important factor to in causinge unlimited proliferation of tumor cells [13]
. Therefore, it
appears to be extremely important forin clinical applications to study whether exogenous hTERT gene
transfection will lead to potential malignant transformation in normal human somatic cells. Karyotype
aAnalysis of chromosome karyotype showed that each hTERT-transfected cell still had 46 chromosomes,
consistent with the number a in a normal somatic cell has. In the experiments of subcutaneous inoculation
in nude mice, the left sides of all nude mice injected with HepG2 cells grew solid tumor masses, while the
right sides injected with SMC and transfected SMCscells were not observeddid not grow any nodules
14

15. or masses, suggesting that telomerase activated by the hTERT gene will not cause infinite cell
proliferation. Tumor formation is simultaneously accompanied by changes in the expression of other
genes, and introduction of hTERT gene into normal somatic cells alone cannot cause malignant
transformation of cells.
Telomerase activity of sensein hTERT-transfected SMCs was significantly increased as compared to
control SMCs. The mitotic index and cell growth curves suggested that SMC-hTERT had stronger higher
cellular growth and proliferation capabilities, as well as a lower incidence of apoptosis than SMC-EGFP
and SMC. This Our data shows that the sense hTERT gene transfection can make humanconfers SMCs
with have a telomerase activity, so that cell proliferation can beis enhanced and cell life-span can be
extended, which is of potential clinical value in the prevention and treatment of organic ED.
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