| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics, Preclinical Pharmacology |
Department of Surgery, Thomas Jefferson University [E. R. S.], Division of Population Science [S. C. L., S. L.], and the Department of Surgery [J. A. R.], Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, and the Department of Molecular and Cellular Biology, The Wistar Institute, Philadelphia, Pennsylvania 19104 [E. R. S., M. H.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
There are a variety of approaches to cancer gene therapy, including 1) local therapy, through direct treatment of the lesion; 2) regional therapy, administered through an artery or vein supplying a given organ(s) or region of the body, or through injection into a body cavity; and 3) systemic therapy, generally administered through venous access. Although limitations exist with each of these approaches, steps are being taken to overcome them. Of the approaches mentioned, direct injection has the advantage of administering a very high dose to the target while minimizing the dose to surrounding tissue.
Cytogenetic analyses of SCCHN have demonstrated a breakpoint on chromosome 11 at 11q13 (1) . The oncogenes int-2, hst-1, and prad1 in the 11q13 amplicon have been reported (2) . Of the three genes, only prad1 (bcl-1, CCND1, cyclin D1) is expressed. Cyclin D1 is a proto-oncogenic regulator of the G1-S checkpoint in the cell cycle that has been implicated in the pathogenesis of several types of cancer, including SCC. Amplification of the cyclin D1 gene is found in 20–50% of SCCs, and the protein is overexpressed in up to 80% of tumors (3 , 4) .
Amplification is only one method by which the protein product can be overexpressed. Increased expression has also been observed due to gene rearrangement (5 , 6) both in parathyroid tumors (11q13 with 11p15) and B cell tumors (11q13 with 14q32). Overexpression of cyclin D1 in cultured cells leads to a more rapid transversion through the G1 phase of the cell cycle and entry into S phase (7 , 8) . Cyclin D1 cooperates with ras (9) and complements a defective Ela adenoviral gene (10) to function as an oncogene.
Other gene products that help regulate the G1 progression include pRb, CDKN2A/p16INK4A, cdk4, and cdk6 (11) . pRb is functional in the vast majority of both primary (12) and immortalized SCCs (13 , 14) . Deletions and mutations in p16 in primary SCCs are uncommon, occurring in 0–20% of cases (15, 16, 17) . Although methylation has been proposed as a major mechanism of p16 gene inactivation in SCCs (18) , other investigators have not found this to be the case (15) . cdk4 and cdk6 gene mutations are rare in cancer of any origin (19) . We elected to study cyclin D1, which is frequently mutated and overexpressed in SCCs, suggesting that alteration of the gene is an important event in SCC development and/or progression.
Cyclin D1 overexpression occurs in preinvasive lesions, and increases from low grade to high grade dysplasia (20) . An increase in intense cyclin D1 expression was observed in the basal and superficial epithelium of oral cavity mucosa from histologically normal:low:high grade (mild:moderate:severe) dysplasias (3:7:19% for basal, 0:0.5:4.6% for superficial epithelium), none of which had a concurrent invasive SCC.
Through the creation of a replication-deficient adenoviral vector containing cyclin D1 in antisense orientation (AS-cyclin D1), we demonstrated in a human xenograft model that AS-cyclin D1 was effective for 1 week in decreasing cyclin D1 expression both in vitro and in vivo, in increasing apoptosis, and in promoting shrinkage of established tumors (21) .
We hypothesized that cyclin D1 overexpression was important not only in
initiation, but also with progression of SCCs. This was addressed using
a two-tiered approach. First, we determined whether cyclin D1
expression increased in advanced clinical (stage III and IV) and
experimental SCCs. Second, we assessed whether treatment with AS-cyclin
D1 led to a persistent reduction in tumor volume. We found that the
incidence of cyclin D1 overexpression increases with disease
progression, that SCC cell lines with CD1 overexpression formed tumors
in 
80 days, whereas cells lacking increased CD1 did not, and
that antisense cyclin D1 therapy leads to a persistent reduction in
tumor volume over a 2- to 4-week follow-up period. These findings
confirm the importance of cyclin D1 in SCC disease progression and
demonstrate the potential efficacy of antisense cyclin D1 gene therapy
to treat SCCs.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Transcomplementing 293 cells (24) , used to grow AS-cyclin D1, were obtained from the Vector Core at the Institute for Human Gene Therapy (University of Pennsylvania, Philadelphia, PA) and grown in DMEM supplemented with 10% FCS. Unless otherwise noted, all tissue culture reagents were purchased from Sigma.
Construction of Replication-Defective Adenoviral Vector.
Construction of an adenoviral vector has been previously described
(25)
. Briefly, A 1.1-kb PCR product with the entire cyclin
D1 open reading frame was inserted into the modified multiple cloning
site (University of Pennsylvania Vector Core) of pSL301 (Invitrogen,
Carlsbad, CA). The resulting plasmid was subcloned into the adenoviral
vector pAd.CMV-Link1 (Vector Core), and the cyclin D1
orientation was determined. pAd.CMV-Link1 was cotransfected into 293
cells containing the E1 gene of Ad5 with adenoviral DNA lacking the E1
and E3 regions (26)
, using calcium phosphate
precipitation. A 10% glycerol shock was then administered, and the
cells were grown with an overlay of 0.8% Bactoagar (Difco Labs,
Detroit, MI). Fresh overlay was added every 3–4 days until plaques
appeared.
Putative plaques were expanded and screened by restriction fragment and Southern blot analyses (27) , a positive AS-cyclin D1 plaque was propagated in 293 cells (28) , the virus was released by freeze-thawing and purified by CsCl gradient centrifugation, and the final plaque-forming units were determined by titration under an agar overlay (29) .
In Vivo Cell Growth.
Cell lines were grown in SCC media to 70–80% confluence and detached
with 0.06% trypsin, the cells counted, and 2 x
106 cells injected s.c. in the dorsal surface of
SCID mice just medial to the right hind limb. Cell lines were
considered tumorigenic if a visible tumor was identified within 80 days
of injection. Established tumors from three lines (A431, SCC 13, and
A253) were evaluated for their response to AS-cyclin D1. After 10–12
replicate tumors formed from each line had grown to a minimum volume of
100 mm3, a single injection of adenovirus of
5 x 108 plaque-forming units and containing
control vector (ß-galactosidase, LacZ) or AS-cyclin D1 in 50 µl of
SCC medium was administered into each tumor using a tuberculin syringe.
Each tumor volume was measured (length x width x height)
for a minimum of 2 weeks and a maximum of 4 weeks after treatment.
Measurements were stopped at 28 days or when control tumors reached
such a large size that euthanasia was required. For statistical
analysis, tumor volumes at baseline and at days 4, 7, 10, 14, 17, 21,
24, and 28 after viral transduction were fitted to an exponential curve
y = A exp(ßt), where ß is the
tumor regression rate estimated for each mouse from the eight
observations. ß values in each group were rank-ordered and analyzed
by the Wilcoxon two-sample (one-sided) procedure to test the hypothesis
of equal rates of tumor regression among groups versus the
alternative that treated tumors regress faster.
Detection of Cyclin D1 by Western Blotting and
Immunohistochemistry.
After growing the cell lines to 70–80% confluence, 100 µg of total
protein was extracted from each sample, separated electrophoretically
under reducing conditions in a discontinuous 12% polyacrylamide gel,
and transferred to a polyvinylidene difluoride membrane. After
transfer, membranes were blocked with 5% nonfat dry milk and probed
with a mouse monoclonal antibody to cyclin D1 (clone HD-11; Santa Cruz
Biotechnology, Santa Cruz, CA) followed by a phosphatase-conjugated
goat antimouse IgG (Jackson ImmunoResearch, West Grove, PA) and the
substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue
tetrazolium. Band intensity was quantified using a personal
densitometer and ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
Thirty formalin-fixed, paraffin-embedded SCCs were cut in 5-µm sections and placed on poly-L-lysine-coated glass slides. After boiling in distilled water for 10 min, slides were incubated with an anticyclin D1 monoclonal Ab (Ab-3; Calbiochem, Cambridge, MA). Horse antimouse IgG was then applied (Vector Laboratories, Burlingame, CA) followed by an avidin-biotin-peroxidase complex (Vector Laboratories) and the chromagen 3',3'-diaminobenzidine (with a counterstain of hematoxylin). Intense immunostain in a given cell was required to consider the cell positive for cyclin D1. One hundred cells in both the basal and germinative layers were counted from two separate areas of the epithelium. The mean number of positive cells was recorded for each specimen. A head and neck carcinoma known to overexpress cyclin D1 and head and neck tumors without primary antibody were used as positive and negative controls.
Statistical Analysis.
The Wilcoxon rank sum test was used to evaluate differences in cyclin
D1 expression in normal, dysplastic, and malignant tissue. A one-sided
test was used to assess significance, given the expectation from our
earlier study (21)
that cyclin D1 expression would
increase with expression of the malignant phenotype.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in cyclin D1 expression (Fig. 1)
in the basal layer from normal
to dysplasia (P = 0.06) and from dysplasia to carcinoma
(P = 0.004). In the germinative layer, cyclin D1
expression increased from dysplasia to carcinoma (P =
0.002) but not from normal to dysplasia. In histologically normal
epithelium adjacent to SCC, the median fraction of cells staining for
cyclin D1 in both the basal and germinative layers was similar to
epithelium from normal donors.
|
|
. A
nonspecific band, routinely seen using this mAb (clone HD-11; Santa
Cruz Biotechnology catalogue), was visualized at 51 kDa. We did not
evaluate A431 by Western blotting because of numerous prior reports
documenting its being both amplified and overexpressed (8
, 32
, 33)
. In the remaining two lines (SCC 12 and SCC 40), a lesser
increase in expression was noted. No significant difference was found
in cyclin D1 expression when comparing cell lines grown with or without
FCS. Of the lines with cyclin D1 overexpression when injected into SCID
mice, 7/8 (88%) formed tumors 80 days after injection, whereas
none of the three lines without cyclin D1 overexpression formed a tumor
within that time period (Table 2)
.
|
|
. Table 3
provides information
regarding tumor growth over a 14-day period from which we can determine
whether tumor growth rate has any influence on the effect of antisense
cyclin D1. AS-cyclin D1 was effective in shrinking A431 (a fast-growing
tumor), A253 (also fast-growing once formed, although taking
approximately 60 days to form a visible tumor in a SCID mouse after
injecting 2 million cells versus 14 days for A431), and SCC
13 (a slow-growing tumor once formed, and taking approximately 3 months
for a visible tumor to form). Although the degree of significance for
the different tumor types varied, this is primarily related to sample
size.
|
visually demonstrates the effect
of AS-cyclin D1 on each of the xenograft tumor types. The Y
axis is different for each tumor type, due to the different growth
rates. Each of the tumors responded to AS-cyclin D1, and the tumor size
remained below baseline for the entire evaluation period (17–28 days).
After treatment with the control (LacZ) vector the tumors continued to
grow, although at different rates.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The response after in vivo treatment of established tumors is promising. Prior reports of in vivo treatment of SCCs using wild-type p53 (34 , 35) document a delay in tumor growth after single injection or stable disease after repeat therapy, rather than demonstrating tumor shrinkage. Our earlier work only addressed tumor response for 1 week after treatment. The current report evaluates the longer term response (up to 4 week) of SCCs to antisense cyclin D1.
Prior reports have demonstrated in vitro (36, 37, 38, 39) and/or ex vivo (38 , 40, 41, 42) inhibition of cell growth with antisense cyclin D1. Our plan was to evaluate the ability of antisense cyclin D1 to inhibit cell growth in established tumors derived from human cancer cells. We chose squamous cell carcinoma, a human tumor system in which the importance of cyclin D1 has been clearly demonstrated. The gene is amplified in up to half of the tumors and the RNA and protein are overexpressed in most cases. Our findings indicate that in vivo transduction with AS-cyclin D1 leads to prolonged tumor shrinkage in a xenograft model of human SCC. Our findings document that: 1) cyclin D1 expression continues to increase with disease progression both in clinical and experimental SCCs and 2) antisense cyclin D1 can shrink established human SCCs, including those from the face, head and neck, and vulva. This suppression persists for at least 2–4 weeks.
We observed regression both in tumors with (A431 and A253) and without (SCC 13) cyclin D1 overexpression. There was not a significant difference in response related to the level of cyclin D1 expression within the tumor. Nonetheless, a greater difference in size (treated versus controls) was observed 2–4 weeks after treatment in tumors overexpressing cyclin D1 (A431: 620% growth versus 62% shrinkage; A253: 324% growth versus 79% shrinkage) than in tumors lacking cyclin D1 overexpression (SCC 13: 36% growth versus 87% shrinkage). Further studies are required to determine whether the level of cyclin D1 expression is an important predictor of response to AS-cyclin D1.
Our model of injecting formed tumors recapitulates more accurately the clinical situation than experiments documenting tumor shrinkage after ex vivo therapy. SCCs of the skin (both facial and vulvar) and the head and neck can be seen in the vast majority of cases. As such, these tumor types are readily accessible to direct injection with antisense cyclin D1. Antisense cyclin D1 treatment could prove to be an appropriate adjunct to currently available therapy, especially when unresectable tumor persists or recurs locally after surgery and radiotherapy. Indeed, significant shrinkage could potentially complement standard treatments such as surgery and radiation.
In a prior report (21) we demonstrated that cyclin D1 induces apoptosis, leading to tumor shrinkage. It is likely that residual viable tumor cells divide and the tumors will eventually regrow. Whether these viable tumor cells transiently undergo G1 arrest or continue to divide in the period immediately following treatment is unclear. Our in vitro experiments in SCC cells did not demonstrate G1 arrest, suggesting that the viable cells continue to divide but that tumor shrinkage occurs as long as cell death exceeds cell replication. Hence, multiple treatments may lead to a further reduction in tumor volume. Prior reports with an adenoviral vector containing wild-type p53 show that multiple doses are more effective than a single treatment (35) . In addition, combination gene therapy may provide a greater reduction in tumor volume.
Although SCC 25 demonstrated increased cyclin D1 expression, it failed to form tumors in immunodeficient mice, contrary to our findings in the other cell lines that had increased cyclin D1 expression. Although it is not entirely clear why findings for SCC 25 are seemingly in conflict with the remaining cell lines, some factors are worth mentioning. First, we found that, although SCC 25 cells grown in serum-free medium did not form tumors in SCID mice, others have found that SCC 25, after in vitro growth in 10% fetal bovine serum, was tumorigenic in nude mice (source: American Type Culture Collection catalogue no. CRL-1628). Another interesting observation is that SCC 25 was one of only two cell lines with cyclin D1 overexpression that, when analyzed, lacked cyclin D1 amplification. The other, SCC 13, formed tumors in SCID mice only after 3 months or more, a longer period of time than for any cell line with cyclin D1 amplification that we studied. Nonetheless, we are not aware of published data demonstrating that the method leading to overexpression (gene amplification versus translocation) affects the tumorigenicity of the cells.
An important question arises as to whether or not the tumor suppressive effect of antisense cyclin D1 is temporary or permanent. As mentioned in "Materials and Methods," measurements were stopped at 28 days or when control tumors reached such a large size that euthanasia was required. Tumors formed from A253 and A431 cells treated with control vector became very large after 2–3 weeks. Although we could have extended the length of time for the cyclin D1-treated tumors, we would not have had a control. We stopped observation of SCC 13 cells after 28 days to be in concordance with the observation time of the other two cell lines. Although we cannot determine from the data available whether the growth suppressor effects of cyclin D1 are transient or permanent, we are concerned that the tumors did not vanish by 21–28 days. In a different tumor system, we are evaluating the possible synergism of two adenoviral vectors with different mechanisms of action. In some of the lesions treated with both vectors, the tumor has vanished within the first 28 days.
In summary, cyclin D1 expression increases with disease progression at all stages of SCC carcinogenesis. Established tumors undergo dramatic shrinkage after treatment with antisense cyclin D1. The response persists for at least 2–4 weeks.
|
FOOTNOTES |
|---|
1 Presented at the 52nd Annual Meeting of the
Society of Surgical Oncology, Orlando, FL, March 4–7, 1999. This work
was supported in part by NIH Grants DE-00380, CA-25874, CA-47159,
CA-76674, and CA-10815. 
2 To whom requests for reprints should be
addressed at: Department of Surgery, Thomas Jefferson University, 1025
Walnut Street, Suite 605, Philadelphia, PA 19107. Phone: (215)
955-4138; Fax: (215) 923-1420; E-mail: edward.sauter{at}mail.tju.edu
3 The abbreviations used are: SCC, squamous cell
carcinoma; FCS, fetal calf serum; SCCHN, SCC of the head and neck;
SCID, severe combined immunodeficiency.
Received 9/23/99; revised 11/18/99; accepted 11/22/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  X. TIAN, A. CHAKRABARTI, N. V. AMIRKHANOV, M. R. ARUVA, K. ZHANG, B. MATHEW, C. CARDI, W. QIN, E. R. SAUTER, M. L. THAKUR, et al. External Imaging of CCND1, MYC, and KRAS Oncogene mRNAs with Tumor-Targeted Radionuclide-PNA-Peptide Chimeras Ann. N.Y. Acad. Sci., November 1, 2005; 1059(1): 106 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. H. Kalish, R. A. Kwong, I. E. Cole, R. M. Gallagher, R. L. Sutherland, and E. A. Musgrove Deregulated Cyclin D1 Expression Is Associated with Decreased Efficacy of the Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Gefitinib in Head and Neck Squamous Cell Carcinoma Cell Lines Clin. Cancer Res., November 15, 2004; 10(22): 7764 - 7774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R CHRISTOPHER, A DHIMAN, J FOX, R GENDELMAN, T HABERITCHER, D KAGLE, G SPIZZ, I G. KHALIL, and C HILL Data-Driven Computer Simulation of Human Cancer Cell Ann. N.Y. Acad. Sci., May 1, 2004; 1020(1): 132 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. TIAN, M. R. ARUVA, P. S. RAO, W. QIN, P. READ, E. R. SAUTER, M. L. THAKUR, and E. WICKSTROM Imaging Oncogene Expression Ann. N.Y. Acad. Sci., December 1, 2003; 1002(1): 165 - 188. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. R. Sauter, U.-C. Yeo, A. von Stemm, W. Zhu, S. Litwin, D. S. Tichansky, G. Pistritto, M. Nesbit, D. Pinkel, M. Herlyn, et al. Cyclin D1 Is a Candidate Oncogene in Cutaneous Melanoma Cancer Res., June 1, 2002; 62(11): 3200 - 3206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Laurance, D. B. Starr, E. F. Michelotti, E. Cheung, C. Gonzalez, A. W. Tam, J. Deikman, C. A. Edwards, and A. J. Bardwell Specific down-regulation of an engineered human cyclin D1 promoter by a novel DNA-binding ligand in intact cells Nucleic Acids Res., February 1, 2001; 29(3): 652 - 661. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
