Skip to main content
  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

AACR logo

  • Register
  • Log in
  • My Cart
Advertisement

Main menu

  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
    • Reviewing
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • Focus on Computer Resources
      • Highly Cited Collection
      • Editors' Picks
      • "Best of" Collection
  • For Authors
    • Information for Authors
    • Author Services
    • Early Career Award
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citations
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

User menu

  • Register
  • Log in
  • My Cart

Search

  • Advanced search
Cancer Research
Cancer Research
  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
    • Reviewing
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • Focus on Computer Resources
      • Highly Cited Collection
      • Editors' Picks
      • "Best of" Collection
  • For Authors
    • Information for Authors
    • Author Services
    • Early Career Award
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citations
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

Tumor Biology

Transforming Growth Factor-β1 Induces Desmoplasia in an Experimental Model of Human Pancreatic Carcinoma

Matthias Löhr, Christian Schmidt, Jörg Ringel, Mario Kluth, Petra Müller, Horst Nizze and Ralf Jesnowski
Matthias Löhr
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Christian Schmidt
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jörg Ringel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mario Kluth
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Petra Müller
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Horst Nizze
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ralf Jesnowski
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI:  Published January 2001
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

Proliferation of fibrotic tissue (desmoplasia) is one of the hallmarks of several epithelial tumors including pancreatic adenocarcinoma. This tissue reaction may be deleterious or advantageous to the host or tumor. In a systematic analysis, we identified two growth factors expressed by human pancreatic carcinoma cells that are positively correlated with the ability to induce fibroblast proliferation both in vitro and in vivo, i.e., transforming growth factor (TGF)-β1 and fibroblast growth factor-2. Here we demonstrate that the overexpression of TGF-β1 induced up-regulation of matrix proteins and growth factors in the TGFβ1-transfected pancreatic tumor cells. Furthermore, transfection of PANC-1 cells induces the same change in fibroblasts in either cocultivation experiments or when they are grown in conditioned medium from TGF-β1-transfected PANC-1 cells. TGF-β1-transfected pancreatic tumor cells induced a rich stroma after orthotopical transplantation in the nude mouse pancreas. The transfer of a single growth factor, TGF-β1, conveys the ability to induce a fibroblast response similar to that seen in desmoplasia in human pancreatic adenocarcinoma. This effect cannot only be attributed to direct effects of TGF-β1 but also results from the up-regulation of several other factors including collagen type I, connective tissue growth factor, and platelet-derived growth factor.

INTRODUCTION

Desmoplasia is a characteristic feature of the growth of some carcinomas (1) . To date, it is not clear whether this process is a mechanism to protect the tumor from the host or represents a defense mechanism by the host (2) , although there are hints that this stroma is beneficial for the tumor (3) . To tackle desmoplasia therapeutically by either supporting or suppressing this development, it becomes necessary to study the etiology and to attribute this feature to either the tumor cells themselves or the host. Desmoplasia is of particular predominance in ductal adenocarcinomas of the pancreas exhibiting a strong stromal reaction (4) . Therefore, pancreatic carcinoma has become a model system to study the interrelation of epithelial tumor cells, matrices, fibroblasts, and growth factors (5, 6, 7, 8) .

Desmoplastic tissue consists of fibroblasts, as the main cellular component, and extracellular matrix proteins (9) . The pancreatic tumor cells themselves are able to produce ECM 3 proteins (10, 11, 12, 13) and interact with ECM by expressing functionally active integrins (6 , 14 , 15) .

To test the hypothesis of desmoplasia induction by a tumor-derived growth factor, we conducted a deductive analysis correlating the ability to induce desmoplasia with the expression of certain growth factors. Furthermore, we reasoned that the overexpression of such a growth factor, e.g., TGF-β1 in a pancreatic tumor cell line known neither to induce desmoplasia nor to express substantial amounts of TGF-β1 and FGF-2, should result in the gain of the ability to induce fibroblast growth and in an induction of desmoplasia in a xenografted nude mouse model by virtue of direct and indirect effects of TGF-β1.

MATERIALS AND METHODS

Cell Culture and Transfection.

AsPC-1, BxPC-3, Capan-1, and PANC-1 cells, all from American Type Culture Collection, were cultivated in DMEM with GlutaMAX I (Life Technologies, Inc.) supplemented with 10% heat-inactivated FCS and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycinsulfate, and 250 ng/ml amphotericin B; Life Technologies, Inc.; Ref. 10 ). Mature human recombinant TGF-β1 was purchased from R&D Systems. Full-length cDNA of TGF-β1 (16) was cut out of pRK5β1E (BamHI) and cloned into the pcDNA3 vector (Invitrogen) under the control of a cytomegalovirus promoter. PANC-1 cells were transfected with this construct or with the empty pcDNA3 plasmid (mock) by calcium phosphate coprecipitation in N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffered saline using standard protocols as described (17) . This plasmid also codes for the neo resistance gene, enabling selection of transfectants with the antibiotic G418 (Sigma; 400 μg/ml). Resistant clones were expanded, and expression of the transfected cDNA was confirmed by Northern blot, Western blot, and ELISA (R&D).

Northern Blot and RT-PCR.

Subconfluent layers of PANC-1/TGF-β1 cells, mock transfected, untransfected PANC-1 cells, and AsPC-1 and BxPC-3 cells were lysed in ice-cold guanidine thiocyanate. RNA preparation was performed as described (18) . Ten μg of total RNA were subjected to standard formamide gel electrophoresis as described. Gels were blotted to nylon membranes (Qiagen) and hybridized with cDNA probes for TGF-β1 (EcoRI/HindIII digest of pcDNA3/TGF-β1), type I collagen (pHCAL1U; Refs. 10 and 19 ), PDGF (Amersham), FGF-2, (20) , and CTGF (21) using the nonradioactive Dig labeling kit (Boehringer Mannheim, Mannheim, Germany). In addition, RT-PCR was performed using published primers for TGF-β1, PDGF-A, type I collagen, and GAPDH. The primers were as follows: TGF-β1 (22) , sense 5′-CAG AAA TAC AGC AAC AAT TCC TGG-3′ and antisense 5′-TTG CAG TGT GTT ATC CCT GCT GTC-3′ (190-bp product); PDGF-A (23) , sense 5′CAG TCA GAT CCA CAG CAT CC-3′ and antisense 5′-AAT GAC CGT CCT GGT CTT GC-3′ (200-bp product); collagen type I (23) , sense 5′-ACG TGA TCT GTG ACG AGA CC-3′ and antisense 5′-AGC AAA GTT TCC TCC GAG GC-3′ (250-bp product); and GAPDH (24) , sense 5′-ACC ACA GTC CAT GCC ATC AC-3′ and antisense 5′-TCC ACC ACC CTG TTG CTG TA-3′ (450-bp product). PCR conditions were the following: denaturing for 30 s at 94°C; annealing for 60 s at 60°C (TGF-β1) or at 64°C (collagen, GAPDH, and PDGF); and extension for 60 s at 72°C. Amplified DNA was sampled after 21, 24, 27, and 30 cycles, and the resulting PCR products for TGF-β1, collagen, and PDGF-A were loaded in the same gel pockets as the GAPDH amplificate.

Reverse Slot Blot.

Expression of genes of several growth factors, receptors, and genes of ECM proteins was investigated by reverse slot blot. For this purpose, plasmid DNA corresponding to 1 μg of cDNA insert was blotted onto a nylon membrane (Qiagen) by use of a slot blot apparatus (Schleicher & Schuell). Hybridization was performed according to standard procedures with a probe obtained by Dig labeling (Boehringer Mannheim) of 7.5 μg of total RNA in a reverse transcription reaction (25 , 26) . Hybrids were detected using the chemiluminescent Dig detection system (Boehringer Mannheim) according to the manufacturer’s instructions.

Cocultivation.

PANC-1/TGFβ1 cells (5 × 104) were seeded onto Transwell inserts (Costar) and were cocultivated with fibroblasts (5 × 104 cells/well) seeded in six-well tissue culture plates. After 7 days of incubation in DMEM/1% FCS, the cells were trypsinized and counted (trypan blue exclusion test). Controls were cocultivated of fibroblasts with fibroblasts, PANC-1, PANC-1 mock transfected, and BxPC-1 cells (American Type Culture Collection). From respective parallel experiments, conditioned media (see below) and RNA (see above) were prepared for subsequent analysis.

Conditioned Media.

PANC-1/TGFβ1 cells were seeded in DMEM/10% FCS. After 2 days of incubation, cells were washed three times with PBS (pH 7.4) to remove any FCS traces and refed with fresh medium containing no FCS. After incubation for another 2 days, the supernatants were collected and filter sterilized. Skin fibroblasts were incubated with serial dilutions of this concentrated medium for 2 days, and induction of proliferation was investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (Boehringer Mannheim). Controls included conditioned media of PANC-1 and mock-transfected PANC-1 cells.

ELISA.

1.3 × 104 cells of TGF-β1-transfected and mock-transfected PANC-1 were plated in six-well plates with DMEM and 10% FCS. After 2 days, cells were grown with DMEM without FCS (transfected cells all of the time with 400μ g/ml G418) for 1, 2, or 3 days, after that the supernatant was collected. TGF-β1 and PDGF were quantified using the Quantikine TGF-β1 and PDGF immunoassays (R&D) according to the instructions of the manufacturer.

Western Blot.

Proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Roche), and blocked for 1 h in Tris-buffered saline (TBS; 10 mm Tris, 10 mm NaCl) containing 1% skim milk and 0.01% Tween 20. After incubation with the primary antibody for 1 h, blots were developed using alkaline phosphatase-labeled secondary antibodies and chemiluminescence (CDP-star; Roche). The following primary antibodies were used in a dilution of 1:1000: PCNA (Santa Cruz; sc-56), TGF-β-1 (sc-146), p21wafI (sc-6246), p-Tyr (sc-7020), Erk 1 (sc-94-G), Erk 2 (sc-1647), and Erk 3 (sc-6268). As detection antibodies, mouse-antigoat immunoglobulin (Dako; 1:5000), rabbit-antimouse immunoglobulin (Dako; 1:5000), and swine-antirabbit immunoglobulin-AP (Dako, 1:5000) were used (27) .

Nuclear Extracts.

Cells were scraped, washed with Tris-buffered saline, resuspended in hypotonic buffer (10 mm HEPES, 10 mm KCl, 1.5 mm MgCl2, and 0.5 mm EDTA), and allowed to swell on ice for 20 min. The nuclei were collected by centrifugation at 12,000 × g for 5 min in a microcentrifuge and analyzed by Western blotting (28) .

Nude Mouse Model.

A suspension of 1 × 106 PANC-1/TGFβ1 cells or mock-transfected PANC-1 cells were injected orthotopically into athymic nude mice (29 , 30) . Nude mice were killed after solid tumors were palpable. Tumors were removed, fixed in 4% formaldehyde, and examined after H&E or Masson-Goldner trichrome staining. Immunocytochemistry was performed as described before with antibodies against type I collagen (1:100; Calbiochem) and fibronectin (1:400; Sigma; Ref. 10 ). Detection was performed using horseradish peroxidase-conjugated rabbit-antimouse and swine-antirabbit IgGs (Dako Diagnostika) as secondary and third antibodies and 3-amino-9-ethylcarbazole as substrate.

RESULTS

Induction of Desmoplasia Is Associated with the Expression of TGF-β1 and FGF-2.

In a deductive analysis on human pancreatic carcinoma cells in vitro and in vivo (6 , 31) , using all of the published information on the expression of various growth factors described in pancreatic carcinoma (22 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) , the stromal reaction (Fig. 1) ⇓ was found to be positively correlated with the expression of TGF-β1 and/or FGF-2 (Ref. 31 ; Table 1 ⇓ ). We therefore chose PANC-1 cells that did not express significant amounts of TGF-β1 as a model for the subsequent experiments investigating the role of TGF-β1 in desmoplasia.

Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

Desmoplastic potential of several human pancreatic adenocarcinoma cell lines upon xenotransplantation on nude mice. Top, tissue culture. Bottom, tumors established on nude mice. Left to right: Panc-1, PaCa-44, Capan-1, and BxPC-3. The two cell lines to the right develop a stroma on the nude mouse. H&E stain,× 250.

View this table:
  • View inline
  • View popup
Table 1

Ability to induce desmoplasia as assessed by induction of stromal tissue upon xenotransplantation in nude mice in human pancreatic carcinoma cell lines

Stable Expression of Functional TGF-β1 in PANC-1 Induces Up-Regulation of Matrix Proteins and Growth Factors.

Expression of the transfected TGF-β1 cDNA in PANC-1/TGF-β1 cells was verified by Northern and Western blots (Figs. 2A ⇓ and 3A) ⇓ . The TGF-β1 protein was released into the culture medium as demonstrated by ELISA of serum-free supernatants; it was native, i.e., inactive, and had to be activated by acidification before quantification.

Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

A, Northern blot of RNA from native Panc-1 cells (Lanes 1 and 2), TGF-β1-transfected Panc-1 cells (Lanes 3 and 4), mock transfected Panc-1 cells (Lanes 5 and 6), and fibroblasts (Lane 7) for TGF-β1 (top) and GAPDH (bottom). Lanes 1, 3, and 5, with FCS; Lanes 2, 4, and 6, without FCS. B, Northern blot of Panc-1/TGF-β1 and Panc-1 for collagen I and ethidium bromide gel (top). Northern blot of Panc-1 +/− FCS (1 + 2); Panc-1/TGFβ1 +/− FCS (2 + 3); Panc-1-mock +/− FCS (5 + 6) and fibroblasts for FGF-2 (bottom). C, reverse slot blot with different cDNA probes hybridized with Dig-labeled cDNA from Panc-1 mock and Panc-1/TGF-β1.

Fig. 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 3.

A, Western blot of whole-cell lysates from mock-transfected Panc-1 cells (Lane 1) and TGF-β1-transfected Panc-1 cells (Lane 2) incubated with antibodies against TGF-β1 (top) and cytokeratin 19 (bottom). B, total cell lysates of pancreatic carcinoma cell lines Panc-1 (Lane 1), mock-transfected Panc-1 (Lane 2), and TGF-β1-transfected Panc-1 (Lane 3) incubated with antibodies against p21waf1 (top) and PCNA (bottom).

In the TGF-β1-transfected PANC-1 cells, the expression of collagen type I was increased (Fig. 2B) ⇓ . Also, PDGF-A was increased (Fig. 2C) ⇓ , whereas the expression of FGF-2 (data not shown), epidermal growth factor, and the α5 integrin subunit (Fig. 2C) ⇓ was similar in TGF-β1-transfected and mock-transfected PANC-1 cells by Northern blot or reverse slot blot.

TGF-β1 inhibits growth by acting on the cell cycle by modulating, for example, p21wafI and PCNA. TGF-β1-transfected PANC-1 cells exhibited a substantial increase in p21wafI expression on the protein level on Western blot of nuclear extracts (Fig. 3B) ⇓ ; on ELISA, p21wafI was 5.4 units/mg protein in untransfected and 16.3 units/mg protein in transfected cells. Conclusively, the transfected cells demonstrated decreased nuclear levels of PCNA on the protein level (Fig. 3B) ⇓ .

TGF-β1-transfected PANC-1 Cells Induce Fibroblast Growth and Up-Regulation of Matrix Proteins and TGF-β1 in Fibroblasts.

Cocultivation of fibroblasts with PANC-1/TGF-β1 cells in the Transwell system led to an increase in proliferation of both the fibroblasts and the tumor cells (Fig. 4A) ⇓ , whereas cocultivation with mock-transfected PANC-1 cells did not exhibit this effect. On the RNA level, induction of collagen type I in the fibroblasts could be demonstrated after incubation with conditioned media from TGF-β1-transfected PANC-1 cells (Fig. 4B) ⇓ ; moreover, an up-regulation of TGF-β1 expression could be demonstrated by RT-PCR under this conditions (Fig. 4B) ⇓ . Although CTGF expression remained unchanged in the PANC-1 cells after TGF-β1 transfection (data not shown), a significant increase in CTGF mRNA was detectable in fibroblasts after cocultivation with TGF-β1-transfected PANC-1 cells (Fig. 4B) ⇓ .

Fig. 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 4.

A, cocultivation of mock-transfected Panc-1 cells and TGF-β1-transfected Panc-1 cells with fibroblasts in the TransWell system. Cultivation of the tumor cells on top in the insert with the fibroblasts in the bottom well or vice versa is shown. The outer of the four columns in each set represent the baseline of tumor cells (left/light gray) and fibroblasts (right/white) grown without cocultivation. The inner columns represent the cell counts for tumor cells (dark gray) and fibroblasts (black) under cocultivation for 3 days. COL I, collagen I. B, Northern blot of RNA from fibroblasts cultivated with and without FCS (Lanes 1 and 2); or with conditioned media from Panc-1 (Lane 3); Panc-1/TGFβ1 (Lane 4) and Panc-1-mock (Lane 5) hybridized for collagen I and 18S rRNA (loading control. Middle: RT-PCR products for TGF-β1 and GAPDH (control) of fibroblasts incubated in DMEM + FCS (Lane 1); or in conditioned media from Panc-1-mock for 1 or 3d (Lanes 2 and 3) and Panc-1/TGF-β1 for 1 or 3 days (Lanes 4 and 5) after 27 (left) and 30 (right) cycles. Bottom, Northern blot for CTGF in fibroblasts after cultivation alone (F) or after cocultivation with mock-transfected (FM) and TGF-β1-transfected (FT) PANC-1 cells (loading control, 18S rRNA).

Incubation of fibroblasts in conditioned media of TGF-β1-transfected PANC-1 cells resulted in more pronounced tyrosine phosphorylation of proteins in fibroblasts than incubation with supernatants from mock-transfected and untransfected PANC-1 cells (Fig. 5 ⇓ , top). Furthermore, mitogen-activated protein kinases were activated as indicated by a mobility shift of Erk 1/2 (Fig. 5 ⇓ , bottom). As mentioned with the tyrosine phosphorylation, the most pronounced phosphorylation of Erk 1/2 and 3 could be demonstrated after incubation with supernatants of PANC-1/TGF-β1 (Fig. 5 ⇓ , bottom). Here, an increase in the activated, i.e., phosphorylated, kinases was evident.

Fig. 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 5.

Induction of tyrosine phosphorylation in fibroblasts after incubation in supernatants from Panc-1, Panc-1 mock, and Panc-1/TGF-β1; Lane 1, control (plain DMEM medium; top). Activation of mitogen-activated protein kinases Erk 1 and Erk 2 after incubation in supernatants (bottom). P, the activated, hence phosphorylated, kinase.

TGF-β1-transfected PANC-1 Cells Induce Desmoplasia with Increase in Matrix Proteins in Vivo.

PANC-1/TGF-β1 transfected cells and mock-transfected cells were injected orthotopically into the nude mouse pancreas. Tumors were harvested after 2 months. The tumors grown from TGF-β1-transfected cells demonstrated an increased desmoplasia surrounding the tumor cells as compared with the mock-transfected cells (Fig. 6) ⇓ . This was evident both on the tumor margin toward the normal mouse pancreas as well as within the tumor. In addition, collagen type I and fibronectin could be detected in increased amounts surrounding the tumor cells (Fig. 6) ⇓ .

Fig. 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 6.

Orthotopic tumors after intrapancreatic injection of TGF-β1-transfected PANC-1 cells (B, D, and F) and mock-transfected PANC-1 cells (A, C, and E) into the nude mouse pancreas. A and B, Masson-Goldner trichrome staining. Immunocytochemistry for collagen type I (C and D) and fibronectin (E and F) is shown.

DISCUSSION

The desmoplastic reaction is one of the morphological hallmarks of several human tumors (1) originating from solid epithelial glands, such as pancreatic adenocarcinoma, that sets it apart from other epithelial tumors. Beside the description and static expression analysis of potential factors, no detailed analysis has been performed to dissect this phenomenon. The pancreatic tumor cells themselves produce matrix proteins (10) and express a variety of integrins (6 , 15) . Furthermore, the expression of growth factors and their receptors has been demonstrated conclusively, however, mostly related to a demonstration of the autocrine growth-promoting effect (35 , 36 , 43) .

To test our hypothesis of a positive correlation of stroma induction and TGF-β1 expression, we successfully transfected the tumor cell line PANC-1 with a TGF-β1 expression vector.

For TGF-β1, it has been suggested that the major regulatory step controlling TGF-β1 activity takes place extracellularly. The same was true for the transfected PANC-1 cells; TGF-β1 was released into the culture medium in a latent, i.e., not activated, state. Recently, it was demonstrated that latent TGF-β1 can bind to and be activated by the αvβ6 integrin (44) . These integrin subunits are also expressed by pancreatic carcinomas (15) , i.e., the PANC-1 cells (6) . Thus, activation of the released TGF-β1 may be accomplished in this way. Expression of TGF-β1 resulted in an up-regulation of the matrix proteins collagen type I and fibronectin in the tumor cells themselves. Furthermore, PDGF expression was increased in the transfected cells. This altered gene expression resulted in several paracrine effects on fibroblasts in cocultivation experiments. We could demonstrate an increase in collagen type I synthesis in the fibroblasts after stimulation with supernatants from TGF-β1-transfected PANC-1 cells. Similarly, the activation of collagen type VII regulatory elements by TGF has been described recently (45) . The fibroblasts themselves produced more TGF-β1 upon stimulation (cocultivation or conditioned media) by the TGF-β1-transfected PANC-1 cells. This is supported by the observation that in pancreatic carcinoma tissue, TGF-β1 is most predominant in the stroma (46) . Furthermore, collagen type I, the most predominant basal membrane matrix protein in pancreatic carcinoma (10) , is also up-regulated, both in the tumor cells themselves and in the fibroblasts upon cocultivation. This up-regulation, however, may only be in part attributed to TGF-β1 itself; it could also be the result of the up-regulation of PDGF-A that has been shown to be a cofactor in TGF-β1-induced collagen type I stimulation (23) . In the fibroblasts, after cocultivation with TGF-β1-transfected PANC-1 cells, CTGF, one of the index TGF-β1 response genes (47) , was increased. Inhibition of CTGF abrogated the TGF-β1-induced collagen gene up-regulation, confirming the pivotal role of this growth factor (48) . As a result of these alterations in gene expression mentioned above, the transfection of TGF-β1 in the pancreatic tumor cell line PANC-1 led to a gain of stromal tissue after orthotopic transplantation in the nude mouse when compared with mock-transfected PANC-1 cells.

The influence of the matrix on signal transduction has long been under debate (49 , 50) . We have shown that a single growth factor, TGF-β1, is capable of conferring the desmoplastic potential to tumor cells not capable of these features. Some of the effects may be attributed to a direct effect of TGF-β1, whereas others, e.g., the up-regulation of collagen type I (51) , may be the result of indirect effects of TGF-β1 intimately associated with the signal transduction pathway involved in TGF-β1 activities.

Acknowledgments

We thank Roland M. Schmid for assistance in subcloning the TGF-β1 plasmid and Thomas Gress (both of University of Ulm, Ulm, Germany) for supplying us with the CTGF plasmid.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 Supported by Grant Lo 431/6 from the Deutsche Forschungsgemeinschaft as part of the special topic program “Matrix in Biology and Medicine” (to M. L.). C. S. acknowledges the support of the Bundesministerium für Bildung und Forschung.

  • ↵2 To whom requests for reprints should be addressed, at Sektion Molekulare Gastroenterologie, Medizinische Klinik IV, Fakultät für Klinische Medizin Mannheim, Universität Heidelberg, Theodor Kutzer Ufer 1-3, D-68135 Mannheim, Germany. Phone: 49-621-383-2900; Fax: 49-381-383-1986; E-mail: matthias.loehr{at}med4.ma.uni-heidelberg.de

  • ↵3 The abbreviations used are: ECM, extracellular matrix; TGF, transforming growth factor; RT-PCR, reverse transcription-PCR; PDGF, platelet-derived growth factor; CTGF, connective tissue growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Dig, digoxygenin; FGF, fibroblast growth factor; Erk, extracellular signal-regulated kinase.

  • Received January 28, 2000.
  • Accepted November 14, 2000.
  • ©2001 American Association for Cancer Research.

References

  1. ↵
    van den Hooff A. Connective tissue changes in cancer. Int. Rev. Connect. Tissue Res., 10: 395-432, 1983.
    OpenUrlPubMed
  2. ↵
    Dvorak H. F. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N. Engl. J. Med., 315: 1650-1659, 1986.
    OpenUrlCrossRefPubMed
  3. ↵
    Sethi T., Rintoul R. C., Moore S. M., MacKinnon A. C., Salter D., Choo C., Chilvers E. R., Dransfield I., Donnelly S. C., Strieter R., Haslett C. Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo. Nat. Med., 5: 662-668, 1999.
    OpenUrlCrossRefPubMed
  4. ↵
    Klöppel G., Lingenthal G., Bülow M. V., Kern H. F. Histological and fine structural features of pancreatic ductal adenocarcinomas in relation to growth and prognosis: studies in xenografted tumours and clinico-histopathological correlation in a series of 75 cases. Histopathol. (Oxf.), 9: 841-856, 1985.
    OpenUrl
  5. ↵
    Longnecker D. S., Jamieson J. D., Asch H. L. Regulation of growth and differentiation in pancreatic cancer. Pancreas, 4: 256-275, 1989.
    OpenUrlPubMed
  6. ↵
    Löhr M., Trautmann B., Peters S., Zauner I., Meier A., Klöppel G., Liebe S., Kreuser E. D. Expression and function of receptors for extracellular matrix proteins in human ductal adenocarcinomas of the pancreas. Pancreas, 12: 248-259, 1996.
    OpenUrlPubMed
  7. ↵
    Ingber D. E., Madri J. A., Jamieson J. D. Neoplastic disorganization of pancreatic epithelial cell-cell relations. Am. J. Pathol., 121: 248-260, 1985.
    OpenUrlPubMed
  8. ↵
    Hall, P. A., and Lemoine, N. R. Models for pancreatic cancer. In: N. R. Lemoine and N. A. Wright (eds.), Cancer Surveys, Vol. 16, The Molecular Pathology of Cancer, pp. 135–155. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1993.
  9. ↵
    Liotta L. A., Rao C. N., Barsky S. H. Tumor cell interaction with the extracellular matrix Liotta L. A. eds. . The Role of Extracellular Matrix in Development, : 357-371, Alan R. Liss, Inc. New York 1984.
  10. ↵
    Löhr M., Trautmann B., Göttler M., Peters S., Zauner I., Maillet B., Klöppel G. Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins. Br. J. Cancer, 69: 144-151, 1994.
    OpenUrlCrossRefPubMed
  11. ↵
    Haberern-Blood C., Liotta L. A., Rao C. N., Kupchik H. Z. Laminin expression by human pancreatic carcinoma cells in the nude mouse and in culture. J. Natl. Cancer Inst., 79: 891-898, 1987.
  12. ↵
    Haglund C., Roberts P. J., Nordling S., Ekblom P. Expression of laminin in pancreatic neoplasms and in chronic pancreatitis. Am. J. Surg. Pathol., 8: 669-676, 1984.
    OpenUrlCrossRefPubMed
  13. ↵
    Tani T., Lumme A., Linnala A., Kivilaakso E., Kiviluoto T., Burgeson R. E., Kangas L., Leivo I., Virtanen I. Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5, and migrate on the newly deposited basement membrane. Am. J. Pathol., 151: 1289-1302, 1997.
    OpenUrlPubMed
  14. ↵
    Weinel R. J., Rosendahl A., Neumann K., Chaloupka B., Erb D., Rothmund M., Santoso S. Expression and function of VLA-α2, -α3, -α5, and α6-integrin receptors in pancreatic carcinoma. Int. J. Cancer, 52: 827-833, 1992.
    OpenUrlCrossRefPubMed
  15. ↵
    Hall P. A., Coates P., Lemoine N. R., Horton M. A. Characterization of integrin chains in normal and neoplastic human pancreas. J. Pathol., 165: 33-41, 1991.
    OpenUrlCrossRefPubMed
  16. ↵
    Wrann M., Bodmer S., Martin R. D., Siepl C., Hofer-Warbinek R., Frei K., Hofer E., Fontana A. T cell suppressor factor from human glioblastoma cells is a 12.5 Kd protein closely related to transforming growth factor-β. EMBO J., 6: 1633-1636, 1987.
    OpenUrlPubMed
  17. ↵
    Jesnowski R., Liebe S., Löhr M. Increasing the transfection efficacy and subsequent long-term culture of resting human pancreatic duct epithelial cells. Pancreas, 17: 262-265, 1998.
    OpenUrlPubMed
  18. ↵
    Sparmann G., Jäschke A., Müller P., Löhr M., Liebe S., Emmrich J. Tissue homogenization as a key step extracting RNA from human and rat pancreatic tissue. Biotechniques, 22: 408-412, 1997.
    OpenUrlPubMed
  19. ↵
    Mäkela J. K., Raassina M., Vuorio E. Human proα1(I) collagen: cDNA sequence for the C-propeptide domain. Nucleic Acids Res., 16: 349 1988.
    OpenUrlFREE Full Text
  20. ↵
    Abraham J. A., Mergia A., Whang J. L., Tumolo A., Friedman J., Hjerrild K. A., Gospodarowicz D., Fiddes J. C. Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science (Washington DC), 545: 545-548, 1986.
    OpenUrl
  21. ↵
    Wenger C., Ellenrieder V., Alber B., Lacher U., Menke A., Hameister H., Wilda M., Iwamura T., Beger H. G., Adler G., Gress T. M. Expression and differential regulation of connective tissue growth factor in pancreatic cancer cells. Oncogene, 18: 1078-1080, 1999.
    OpenUrl
  22. ↵
    van Laethem J. L., Resibois A., Rickaert F., Deviere J., Gelin M., Cremer M., Robberecht P. Different expression of transforming growth factor β1 in pancreatic ductal adenocarcinoma and cystic neoplasms. Pancreas, 15: 41-47, 1997.
    OpenUrlPubMed
  23. ↵
    Halloran B. G., So B. J., Baxter B. T. Platelet-derived growth factor is a cofactor in the induction of 1 α(I) procollagen expression by transforming growth factor-β 1 in smooth muscle cells. J. Vasc. Surg., 23: 767-773, 1996.
    OpenUrlCrossRefPubMed
  24. ↵
    Ning Y., Roschke A., Christian S., Lesser J., Sutcliffe J. S., Ledbetter D. H. Identification of a novel paternally expressed transcript adjacent to snRPN in the Prader-Willi syndrome critical region. Genome Res., 6: 742-746, 1996.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    von Heimendahl G., Gebhardt E., Dingermann T. Proto-oncogene activation in surgical specimens of rectal carcinoma. Anticancer Res., 8: 805-812, 1988.
    OpenUrlPubMed
  26. ↵
    Jesnowski R., Müller P., Schareck W., Liebe S., Löhr M. Immortalized pancreatic duct cells in vitro and in vivo. Ann. NY Acad. Sci., 880: 50-65, 1999.
    OpenUrlCrossRefPubMed
  27. ↵
    Schmidt C., Pommerencke H., Dürr F., Nebe B., Rychly J. Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins. J. Biol. Chem., 273: 5081-5085, 1998.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Tilbrook P. A., Bittorf T., Callus B., Busfield S. J., Spadaccini A., Ali M., Ingley E., Klinken S. P. Regulation of the erythropoietin receptor and involvement of JAK2 in differentiation of J2E erythroid cells. Cell Growth Differ., 7: 511-520, 1996.
    OpenUrlAbstract
  29. ↵
    Fu X., Guadagni F., Hoffman R. M. A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens. Proc. Natl. Acad. Sci. USA, 89: 5645-5649, 1992.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Reyes G., Villanueva A., Garciá C., Sancho F. J., Piulats J., Lluís F., Capellá G. Orthotopic xenografts of human pancreatic carcinomas acquire genetic aberrations during dissemination in nude mice. Cancer Res., 56: 5713-5719, 1996.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Löhr M. Stromal-epithelial interactions in pancreatic carcinomas Lemoine N. R. Neoptolemos J. eds. . Cell Biology of Pancreatic Cancer, : 18-35, Blackwell Science London 1996.
  32. ↵
    Korc M., Meltzer P., Trent J. Enhanced expression of epidermal growth factor receptor correlates with alterations of chromosome 7 in human pancreatic cancer. Proc. Natl. Acad. Sci. USA, 83: 5141-5144, 1986.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Leung H. Y., Hughes C. M., Klöppel G., Williamsons R. C. N., Lemoine N. R. Localization of expression of fibroblast growth factors and their receptors in pancreatic adenocarcinoma by in situ hybridization. Int. J. Oncol., 4: 1219-1223, 1994.
    OpenUrlPubMed
  34. ↵
    Estival A., Clerc P., Vayesse N., Tam J. P., Clemente F. Decreased expression of transforming growth factor α during differentiation of human pancreatic cancer cells. Gastroenterology, 103: 1851-1859, 1992.
    OpenUrlPubMed
  35. ↵
    Leung H. Y., Gullick W. J., Lemoine N. R. Expression and functional activity of fibroblast growth factors and their receptors in human pancreatic cancer. Int. J. Cancer, 59: 667-675, 1994.
    OpenUrlPubMed
  36. ↵
    Kornmann M., Beger H. G., Korc M. Role of fibroblast growth factors and their receptors in pancreatic cancer and chronic pancreatitis. Pancreas, 17: 169-175, 1998.
    OpenUrlPubMed
  37. ↵
    Friess H., Yamanaka Y., Büchler M., Ebert M., Beger H. G., Gold L. I., Korc M. Enhanced expression of transforming growth factor β isoforms in pancreatic cancer correlates with decreased survival. Gastroenterology, 105: 1846-1856, 1993.
    OpenUrlPubMed
  38. ↵
    Barton C., Hall P. A., Hughes C. M., Gullick W. J., Lemoine N. R. Transforming growth factor α and epidermal growth factor in human pancreatic cancer. J. Pathol., 163: 111-116, 1991.
    OpenUrlCrossRefPubMed
  39. ↵
    Beauchamp R. D., Lyons R. M., Yang E. Y., Coffey R. J., Moses H. L. Expression of and response to growth regulatory peptides by two human pancreatic carcinoma cell lines. Pancreas, 5: 369-380, 1990.
    OpenUrlCrossRefPubMed
  40. ↵
    Kalthoff H., Roeder C., Humburg I., Thiele H. G., Greten H., Schmiegel W. Modulation of platelet-derived growth factor A- and B-chain/c-sis mRNA by tumor necrosis factor and other agents in adenocarcinoma cells. Oncogene, 6: 1015-1021, 1991.
    OpenUrlPubMed
  41. ↵
    Kalthoff H., Roeder C., Gieseking J., Humburg I., Schmiegel W. Inverse regulation of human ERBB2 and epidermal growth factor receptors by tumor necrosis factor α. Proc. Natl. Acad. Sci. USA, 90: 8972-8976, 1993.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Schmiegel W., Roeder C., Schmielau J., Rodeck U., Kalthoff H. Tumor necrosis factor α and the epithelial growth factor receptor in human pancreatic cancer cells. Proc. Natl. Acad. Sci. USA, 90: 863-867, 1993.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Lemoine N. R., Leung H. Y., Barton C. M., Hughes C. M., Klöppel G., Gullick W. J. Autocrine growth control of pancreatic cancer. Int. J. Pancreatol., 14: 69-70, 1993.
  44. ↵
    Munger J. S., Huang X., Kawakatsu H., Griffiths M. J. D., Dalton S. L., Wu J., Pittet J. F., Kaminski N., Garat C., Matthay M. A., Rifkin D. B., Sheppard D. The integrin αvβ6 binds and activates latent TGF-β 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell, 96: 319-328, 1999.
    OpenUrlCrossRefPubMed
  45. ↵
    Rodeck U., Nishiyama T., Mauviel A. Independent regulation of growth and SMAD-mediated transcription by transforming growth factor β in human melanoma cells. Cancer Res., 59: 547-550, 1999.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Satoh K., Shimosegawa T., Hirota M., Koizumi M., Toyota T. Expression of transforming growth factor β1 (TGFβ1) and its receptors in pancreatic duct cell carcinoma and in chronic pancreatitis. Pancreas, 16: 468-474, 1998.
    OpenUrlPubMed
  47. ↵
    Geng M. M., Ellenrieder V., Wallrapp C., Müller-Pilasch F., Sommer G., Adler G., Gress T. M. Use of representational difference analysis to study the effect of TGFβ on the expression profile of a pancreatic cancer cell line. Genes Chromosomes Cancer, 26: 70-79, 1999.
    OpenUrlCrossRefPubMed
  48. ↵
    Duncan M. R., Frazier K. S., Abramson S., Williams S., Klapper H., Huang X., Grotendorst G. R. Connective tissue growth factor mediates transforming growth factor β-induced collagen synthesis: down-regulation by cAMP. FASEB J., 13: 1774-1786, 1999.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Bissell M. J., Hall H. G., Parry G. How does the extracellular matrix direct gene expression?. J. Theor. Biol., 99: 31-68, 1982.
    OpenUrlCrossRefPubMed
  50. ↵
    Bissell M. J., Barcellos-Hoff M. H. The influence of extracellular matrix on gene expression: is structure the message?. J. Cell Sci. (Suppl.), 8: 327-343, 1987.
    OpenUrlPubMed
  51. ↵
    Sparmann G., Merkord J., Jäschke A., Nizze H., Jonas L., Löhr M., Liebe S., Emmrich J. Pancreatic fibrosis in experimental pancreatitis induced by dibutylin dichloride. Gastroenterology, 112: 1664-1672, 1997.
    OpenUrlCrossRefPubMed
  52. ↵
    Kalthoff, H., Löhr, M., Roeder, C., and Schmiegel, W. Das Pankreaskarzinom. Zellbiologie, Matrixproteine und Wachstumsregulation. In: G. Adler, U. R. Fölsch, J. Mössner, and M. V. Singer (eds.), Erkrankungen des exkretorischen Pankreas, Ed. 2, pp. 385–404. Jena: Fischer, 1995.
View Abstract
PreviousNext
Back to top
Cancer Research: 61 (2)
January 2001
Volume 61, Issue 2
  • Table of Contents

Sign up for alerts

View this article with LENS

Open full page PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Cancer Research article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Transforming Growth Factor-β1 Induces Desmoplasia in an Experimental Model of Human Pancreatic Carcinoma
(Your Name) has forwarded a page to you from Cancer Research
(Your Name) thought you would be interested in this article in Cancer Research.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Transforming Growth Factor-β1 Induces Desmoplasia in an Experimental Model of Human Pancreatic Carcinoma
Matthias Löhr, Christian Schmidt, Jörg Ringel, Mario Kluth, Petra Müller, Horst Nizze and Ralf Jesnowski
Cancer Res January 1 2001 (61) (2) 550-555;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Transforming Growth Factor-β1 Induces Desmoplasia in an Experimental Model of Human Pancreatic Carcinoma
Matthias Löhr, Christian Schmidt, Jörg Ringel, Mario Kluth, Petra Müller, Horst Nizze and Ralf Jesnowski
Cancer Res January 1 2001 (61) (2) 550-555;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • INTRODUCTION
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF
Advertisement

Related Articles

Cited By...

More in this TOC Section

  • Abstract 6119: RNAi rat models for drug discovery
  • Abstract 3834: Histone methyltransferase SET8 is regulated by miR-192/-215 and induces oncogene-induced senescence via p53-dependent DNA damage in human gastric carcinoma cells
  • Abstract 3788: CircHMGCS1 interacts with RNA binding protein HuR and maintains stem-like cells in gliomas
Show more Tumor Biology
  • Home
  • Alerts
  • Feedback
  • Privacy Policy
Facebook  Twitter  LinkedIn  YouTube  RSS

Articles

  • Online First
  • Current Issue
  • Past Issues
  • Meeting Abstracts

Info for

  • Authors
  • Subscribers
  • Advertisers
  • Librarians

About Cancer Research

  • About the Journal
  • Editorial Board
  • Permissions
  • Submit a Manuscript
AACR logo

Copyright © 2021 by the American Association for Cancer Research.

Cancer Research Online ISSN: 1538-7445
Cancer Research Print ISSN: 0008-5472
Journal of Cancer Research ISSN: 0099-7013
American Journal of Cancer ISSN: 0099-7374

Advertisement