An in vitro model of the early genetic events in multistage carcinogenesis of malignant insulinoma

Masa Katic, Mirko Hadzija, Mercedes Wrischer1 and Kresimir Pavelic2

Division of Molecular Medicine and
1 Division of Molecular Genetics, Rudjer Boskovic Institute, Bijenicka 54, 10 000 Zagreb, Croatia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to establish an in vitro model to confirm earlier observations on the role of the myc/ras oncogenes as promoting factors in the process of normal Langerhans islet ß cell transformation. For that purpose we infected primary mouse Langerhans islets with a recombinant retrovirus containing the v-H-ras and v-myc oncogenes, before or after treatment with transforming growth factor {alpha} (TGF{alpha}). Normal Langerhans islets, when grown in culture, are viable for 2–3 weeks. After treatment with TGF{alpha}, viability was extended by 10 days, following which islets disintegrated. Langerhans islets transformed with v-H-ras and v-myc became immortal and insulin negative. Single infected ß cells, liberated from a primary islet into the surrounding medium, gave rise to neo islet formation. Moreover, single infected ß cells were able to grow and divide, even without fibroblast support. These results indicate that the myc and ras oncogenes are sufficient for commencement of ß cell transformation and, therefore, could represent `early events' in the multistep carcinogenesis of insulinomas.

Abbreviations: c.f.u., colony forming units; EGF-R, epidermal growth factor receptor; FCS, fetal calf serum; HeBS, HEPES-buffered saline pH 7.05; PBS, phosphate-buffered saline; TGF{alpha}, transforming growth factor {alpha}


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carcinogenesis is a multistep process in which normal cells acquire full malignancy by the successive activation of dominant oncogenes and/or inactivation of tumor suppressor genes (13). Considerable evidence shows that at least two oncogenes, representing different functional classes (nuclear and cytoplasmic oncoproteins), are needed for full transformation of a cell (35). In agreement with this, several human tumors and tumor cell lines have been found to contain two activated oncogenes, representing one from each of the ras and myc gene families (68).

Pancreatic cancer is the fifth leading cause of cancer death in Western society (9). Early diagnosis of that cancer remains elusive and patients often present with locally advanced or metastatic disease (10). Endocrine tumors of the pancreas are relatively rare. Most of them grow slowly and are generally associated with a long-term prognosis (11). Metastases occur in 5–10% of patients with insulinoma, concomitant with poor prognosis and a reported 5 year survival rate of 30–40% (12,13).

Relatively little is known about the role of oncogenes and tumor suppressor genes in malignant metastatic insulinomas, probably due to the rarity of this tumor. The results of our previous work (14) suggest that the accumulation of genetic alterations, including c-myc and transforming growth factor {alpha} (TGF{alpha})/epidermal growth factor receptor (EGF-R) overexpression, c-K-ras point mutation (14,15) and overexpression of p53 mutant protein (14,16), may contribute to tumor development and/or progression from benign to malignant metastatic insulinoma. Mutation of codon 61 in metastatic insulinoma and increased level of N-Ras protein (from B cell hyperplasia to malignant insulinoma) was also found (15,16), but without the data about differential expression of N-ras and H-ras genes.

The aim of this study was to establish an in vitro model to confirm these observations and to prove that the myc/ras oncogenes are promoting factors in neoplastic transformation of cells.

Primary rodent and human Langerhans islets isolated from adults show limited growth ability in vitro because the majority of cells are in the G0 phase of the cell cycle. Since we used a retroviral construct in our experiments this was a major obstacle to studying `early genetic events' in in vitro carcinogenesis of insulinomas. For that reason we chose newborn mice Langerhans islets, ß cells from which divide in culture for 3–4 weeks (17).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Production of recombinant retrovirus and infection of Langerhans islets
The PA317 amphotropic packaging cell line, as well as NIH 3T3 fibroblasts and Langerhans islets, were maintained in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY) with 1000 mg/l glucose, 10 mM HEPES (Sigma, St Louis, MO), 2 mM L-glutamine (Sigma), 100 U/ml penicillin (Pliva, Zagreb, Croatia), 100 µg/ml streptomycin (Pliva) and 10% fetal calf serum (FCS) (Gibco BRL).

The retroviral construct Zipras/myc9, a member of the Zip class of retroviral constructs, contains the v-H-ras oncogene from Harvey sarcoma virus and the v-myc oncogene from MC12 virus.

Transfection of PA317 cells with the Zipras/myc9 retroviral construct was performed by the CaPO4 transfection method (18,19). Aliquots of 1.64x106 cells were plated in two tissue culture bottles (250 ml) (Greiner, Frickenhausen, Germany) 1 day before transfection with 10 µg of the retroviral construct mixed with HEPES-buffered saline, pH 7.05 (HeBS) and CaCl2. The HeBS–DNA precipitate (45 min at room temperature) was added to the cells (20 min at room temperature) and after addition of medium they were placed for 4 h at 37°C, 5% CO2. After incubation, the medium was aspirated, HeBS/glycerol was added and the cells were placed in the incubator for another 3.5 min. Cells were rinsed twice with medium and 5 ml of medium supplemented with FCS (10%) were added. One day later, transiently produced virus was collected, 10x concentrated (Amicon, Beverly, MA) and filtered through a 0.45 µm filter (Sarstedt, Nümbrecht, Germany). Virus stock concentration was determined [colonies in soft agar in colony forming units (c.f.u.)] (19) by infection of NIH 3T3 fibroblasts with the addition of 8 µg/ml polybrene (Sigma).

Primary Langerhans islets were isolated from newborn (at the day of delivery) CBA/HZgr mice by collagenase digestion and purified as described previously (20). The day before retroviral infection, Langerhans islets were plated in a 3 cm culture dish (Greiner). On the day of infection the medium was replaced with 2 ml of medium containing from 2.4–3.6x103 c.f.u., supplemented with 8 µg/ml polybrene. Infection was continued for 3 h at 37°C, 5% CO2, following which the medium was replenished. In some experiments, Langerhans islets were treated with TGF{alpha} (Sigma), at a final concentration of 1 µg/ml, for the next 3 months. In other experiments Langerhans islets were infected not earlier than 3 weeks after seeding. During this period they were maintained in medium supplemented with TGF{alpha} (1 µg/ml).

Polymerase chain reaction (PCR)
PCR analysis was used to confirm the presence of the Zipras/myc9 retroviral construct in infected Langerhans islets (ß cells). For DNA isolation, Langerhans islets were trypsinized, washed with phosphate-buffered saline (PBS) three times and resuspended in extraction buffer (10 mM Tris–HCl, pH 8.0, 0.1 M EDTA, pH 8.0, 0.5% SDS). After overnight incubation at 37°C with 100 µg/ml proteinase K (Sigma), a 1/3 vol of 5 M NaCl was added and the mixture was incubated at 4°C for 15 min. After a 15 min centrifugation at 13 000 g the supernatant was transferred to another tube and 2 vol of 100% ethanol (–20°C) were added. The DNA pellet appeared after overnight incubation at –20°C and 20 min centrifugation at 13 000 g. The pellet was washed with 70% ethanol, dried and resuspended in TE buffer (10 mM Tris–HCl, pH 8.0, 0.1 M EDTA, pH 8.0).

Primers for the PCR reaction were designed for the U5 region of Moloney murine leukemia retrovirus LTR sequence (Table IGo).


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Table I. Primers, their position and direction for U5 retroviral sequence
 
PCR reactions were carried out in a 50 µl reaction volume containing 500 ng of template, 8 pmol of U5A and U5B primers, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 500 µM each dNTP. One unit of Taq polymerase and paraffin oil were added after 3 min denaturation at 96°C (PCR-DNA Thermal Cycler type 480; Perkin Elmer-Cetus, Norwalk, CT). The amplification was performed for 40 cycles of 96°C for 1 min (denaturation), 55°C for 45 s (annealing) and 72°C for 1 min (elongation). Final extension was done at 72°C for 10 min. The PCR product (215 bp) was visualized in ethidium bromide stained 2.5% agarose gel.

Immunocytochemistry
For immunocytochemical staining, cells were washed twice with PBS and fixed in situ in a culture dish (10x35 mm; Greiner) with 3 ml ethanol/10% TCA for 1 h at –20°C. After fixation, cells were washed with PBS once more. Immunocytochemical detection of c-Myc (Ab-1; Oncogene Science, Cambridge, MA), c-H-Ras (Ab-1; Oncogene Science) and insulin (mAb-I5; Rudjer Boskovic Institute, Zagreb, Croatia) was performed by incubation with monoclonal antibodies for 30 min at 4°C. After an additional 30 min incubation with goat anti-mouse FITC-labeled secondary antibodies at 4°C, staining was monitored with a fluorescence microscope (Binolux-Zetopan, Reichart, Wien, Austria). Negative controls were treated in the same way, except that the primary antibodies were omitted.

The localization and level of specific immunostaining for control and transformed cultures were evaluated semi-quantitatively in the whole culture dish. The relative level and localization of specific immunostaining were judged. The immunostaining was evaluated semi-quantitatively and denoted as weak (+), moderate (++) or strong (+++).

Electron microscopy
For electron microscopy analysis infected and control Langerhans islets were trypsinized, extensively washed with PBS and fixed in 0.2 M cacodylate buffer (pH 7.2) with 8% glutaraldehyde, followed by a PBS wash for 2 h. Islets were covered with 2% agarose and post-fixed in 1% osmium tetroxide.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture of Langerhans islets
Normal non-infected Langerhans islets survived in culture for 14–21 days. After that period the islets disintegrated and only fibroblasts remained. When TGF{alpha} was added to the culture medium the disintegration of normal islets was postponed for 10 days. Langerhans islets transformed with the oncogenes ras and myc became immortal when grown in culture. Three months after seeding, when the islets were used for further experiments (not described in this paper), they were still viable. A week after infection significant cell proliferation, particularly at the margins of the islets, was observed (Figure 1Go). During the course of cultivation (usually 2 months after infection) some of these cells, liberated from the primary islet into the surrounding medium, gave rise to new islet (so-called neo islet) formation (Figure 2Go). Since fibroblasts inevitably carried over in the isolation of Langerhans islets, in all the aforementioned experiments we were in fact dealing with mixed cultures. It is important to emphasize that Langerhans islets cannot grow in culture without fibroblasts. When single infected ß cells, separated from fibroblasts, were seeded in culture they changed phenotypically and became dark granulated cells (Figure 3Go). Although they retained proliferative capacity, they were not able to form new islets, even after addition of fibroblasts to the dish.



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Fig. 1. Transformed Langerhans islet a week after infection. Significant proliferation of cells is visible at the margins of the islet. 120x.

 


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Fig. 2. Neo islet formation 2 months after infection of Langerhans islets with the recombinant retrovirus. 120x.

 


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Fig. 3. Single infected ß cells, separated from fibroblasts, change phenotypically and become dark and granulated. 120x.

 
Electron microscopic analysis of normal and infected Langerhans islets revealed their ultrastructural differences. While normal ß cells contained numerous insulin granula in their cytoplasm (Figure 4Go), transformed ß cells contained only a few. Transformed cells contained many different membranous structures in their cytoplasm (Figure 5Go) which were not seen in normal ß cells.



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Fig. 4. Electron micrograph of normal newborn mouse ß cell. Many insulin granula (arrow) are visible in the cytoplasm of normal ß cells. 10 000x.

 


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Fig. 5. Electron micrograph of transformed ß cell. There are no insulin granula, but many different membranous structures are visible in the cytoplasm of ß cells transformed with oncogenes ras and myc. 10 000x.

 
PCR amplification of U5 LTR retroviral DNA was used to assay the presence of the retroviral construct in the infected Langerhans islets (Figure 6Go) as well as in the single infected ß cells (data not shown). A specific U5 215 bp long fragment was always present in DNA from the infected Langerhans islets and single ß cells, whereas it was absent in the non-infected ß cells.



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Fig. 6. PCR amplification of U5 region of retroviral LTR sequences. Lanes 1 and 7, DNA marker VIII (Boehringer-Mannheim, Mannheim, Germany); lane 2, retroviral construct Zipras/myc9; lane 3, NIH 3T3 fibroblasts; lane 4, PA317 packaging cells; lane 5, normal Langerhans islets DNA; lane 6, transformed Langerhans islets DNA. In lanes 2, 4 and 6 there is the PCR product of 215 bp.

 
Addition of TGF{alpha}, either before or after infection, had no influence on the cell transformation process.

Immunocytochemistry
The results of immunocytochemical staining of infected Langerhans islet cultures (including fibroblasts) are summarized in Table IIGo. These are not absolute numbers since there was not the same ratio of fibroblasts and ß cells/Langerhans islets in each culture dish examined. However, taking that into account, immunocytochemical staining showed altered expression of c-Myc, c-H-ras and insulin in transformed islets compared with the control ones. Forty-seven per cent of the cells in transformed islet cultures showed positive staining for c-Myc (~80% of islet cells and ~15% of fibroblasts) and 15% for Ras (>90% of ß cells and only a few fibroblasts), whereas only 11 and 7%, respectively, of all cells were positive in normal culture (Figures 7 and 8GoGo). Insulin expression was found in only 10% of the cells in infected Langerhans islets, whereas 85% of the cells showed strong insulin positivity in normal islets (Figure 9Go). Fibroblasts showed no positivity for insulin, in either normal or transformed culture.


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Table II. Immunocytochemical detection of c-Myc, c-H-ras and insulin in normal and transformed Langerhans islets cultures
 



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Fig. 7. Immunocytochemical staining of normal (A) and infected (B) Langerhans islet culture for the expression of c-Myc. Normal Langerhans islets, only a few c-Myc-positive cells; infected Langerhans islets, ~80% c-Myc-positive cells. 360x.

 





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Fig. 8. Immunocytochemical staining of normal (A and B) and infected (C and D) Langerhans islet culture for the expression of c-H-Ras. Light blue, round cells in (A) and (C) present ß cells, whether single or within neo islets. Normal Langerhans islets (B) have only a few c-H-Ras-positive cells (weak immunostaining, arrow); infected Langerhans islets and neo islets in (D) (arrows) have >90% of cells positive for Ras. 120x.

 



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Fig. 9. Immunocytochemical staining of normal (A) and infected (B) Langerhans islets culture for the expression of insulin. Normal ß cells show strong positivity for insulin; infected Langerhans islets have 11% positive cells (weak immunostaining) (arrow). 120x.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our previous work (14) showed that the accumulation of several genetic alterations contributes to malignant insulinoma development. Overexpression of myc and TGF{alpha}/EGF-R as well as point mutation in the K-ras gene (and increased levels of other members of the Ras family of proteins; 21) appear relatively early, whereas other types of mutations occur later in the course of tumor progression.

In the present study we have investigated the role of the myc and ras oncogenes as well as the growth factor TGF{alpha} in in vitro transformation of normal mouse Langerhans islets. This was achieved by introducing the ras and myc oncogenes into newborn mouse Langerhans islets in vitro in combination with TGF{alpha}.

As has already been pointed out, Langerhans islet cultures, when isolated, consist mostly of Langerhans islets and only a small portion of fibroblasts. Later only fibroblasts remained. Islets disintegrate and after some time ß cells disappear from the culture by apoptosis (21). The pancreas of newborn mice has ~40 Langerhans islets, each of which consists of ~2000 cells (>70% ß cells). Usually 15 newborn mice were used in each experiment. We did not measure the incidence of infection, but knowing the approximate number of cells and viruses used for each experiment, theoretically 1–2% of cells could have been infected with the retroviral particles. Since there were many more islet cells dividing than fibroblasts at the time of the infection, the possibility of their infection was much higher.

A basic parameter indicative of transformation in any tissue is its lifespan when grown in culture. The lifespan of normal mouse Langerhans islets when grown in culture was ~2–3 weeks, after which time they disintegrated and disappeared from the culture by apoptosis, as described earlier (21). However, Langerhans islets infected with the retroviral ras/myc construct became immortal when grown in culture. The cells retained the capacity to proliferate, which resulted in neo islet formation. These neo islets are not really functional units, but represent aggregates consisting of infected ß cells, which is confirmed by immunocytochemical staining. It has been shown that these cells were strongly positive for Myc as well as Ras oncoproteins, while fibroblasts mostly showed only Myc positivity. Since fibroblasts have a capacity for longer life in culture than ß cells, we believe that Myc positivity is a reflection of their normal proliferation and not a consequence of infection. The fact that the majority of Myc-positive cells in control cultures were fibroblasts (4 days after plating) contributes to that assumption. However, if some fibroblasts were also infected it would be possible that they influenced Langerhans islet transformation. Although we cannot differentiate between expression of v-myc and c-myc by immunocytochemical staining, we can be sure that Myc positivity of ß cells is due to infection of these cells by comparing immunocytochemical results for control and transformed cultures.

Our earlier finding of c-myc overexpression in benign insulinoma (14,15) indicates that the immortalization of ß cells was due to the action of the myc, but not the ras, oncogene. Similar results were shown for chemically transformed A31 cells (BPA31 and DA31 cells) (22). Myc protein was often found in malignant tissues, which is connected with intensive proliferation of these cells. Therefore, we assume that immortalization of ß cells in vitro was also due to the action of the myc oncogene.

Since normal Langerhans islets when treated with TGF{alpha} alone did not undergo any changes visible under light microscopy which would indicate cell transformation, TGF{alpha} does not act in the process of Langerhans islets transformation earlier than c-myc. On the other hand, fibroblasts did undergo visible phenotypic changes (becoming more elongated with more extensions). This is consistent with previously described results where transformation of the exocrine parts of the pancreas (hyperplasia of acinar cells) and fibroblasts (fibroplasia), but not Langerhans islets, were found in transgenic mice (with the TGF{alpha} transgene under control of the elastine promoter) (23). Considering the aforementioned results it seems that TGF{alpha} could act in a tissue-specific manner in the process of neoplastic transformation.

Additional proof of Langerhans islet transformation came from the behaviour of single infected ß cells. When separated from Langerhans islets and seeded in culture without fibroblasts they became dark and granulated, but were still able to divide. However, they were not able to form islets. We assume that they became independent of some (growth) factors secreted by fibroblasts.

Infected ß cells became insulin negative and contained many cytoplasmic membranous structures, different from those that represent the retroviral genome itself (24). Decreased production of insulin and the lack of insulin granula in the cytoplasm of transformed ß cells could be due to the action of the ras oncogene introduced into these cells. The major intracellular substrates of the insulin receptor tyrosine kinase are insulin receptor substrates 1 and 2 (25,26). Upon ligand stimulation these molecules rapidly become phosphorylated on multiple tyrosine residues and bind a variety of signalling proteins via their src homology-2 domain (2729). One of the most recent results demonstrated that insulin treatment of Chinese hamster ovary cells expressing the human insulin receptor results in derepression of the Rap1 inhibitory function on Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade (30). This suggests that insulin controls ras signalling as well. In this respect, permanently activated ras in transformed cells, since it acts downstream of the insulin receptor, could decrease synthesis of insulin by a feedback mechanism. One of the most striking examples of ras oncogene activation in human tumors is exocrine pancreatic adenocarcinoma. Over 90% of this tumor type contain mutated ras genes (31,32). Mutated ras genes, together with myc overexpression and weak or moderate insulin expression, were also found in malignant insulinoma of human origin. The phenotype of the transformed cells presented in this paper bears a striking resemblance to the in vivo situation. Therefore, it may represent an in vitro phenotype positioned toward malignancy.

Our results suggest that cooperation of activated ras and myc oncogenes can contribute to the initiation of cancerogenesis of insulinoma. This is consistent with previous results on the concomitant action of the ras and myc oncogenes, regardless of whether they were introduced into normal cells (33), reconstituted organs (34) or as transgenes in vivo (35). Additional mutations are probably necessary for full malignancy of ß cells.

Addition of TGF{alpha} to the medium of infected Langerhans islets did not cause any further changes in transformation of ß cells. There are two possible reasons for this result. It is known that TGF{alpha} acts by binding to the EGF-R. In malignant insulinoma overexpression of EGF-R was found to be concomitant with overexpression of TGF-{alpha} (14). Also, others have found that TGF{alpha} could transform immortal fibroblasts only with concomitant overexpression of EGF-R (36). However, since we did not follow the expression of EGF-R in our model system, we do not know whether it was overexpressed. If it was not, an increased amount of TGF{alpha} could not influence ß cells. On the other hand, elevated expression of TGF{alpha} has been consistently associated with the pathological process of neoplastic transformation. It was first identified in medium conditioned by retrovirus-transformed fibroblasts (37). TGF{alpha} expression can also be induced in cultured cells by transformation with several viral and cellular oncogenes or by treatment with carcinogens or tumor promoting factors (38,39). Our results suggest that introduction of the myc and ras oncogenes into ß cells could cause endogenous overexpression of TGF{alpha}/EGF-R. In that case exogenous addition of TGF{alpha} could not further affect these cells.

In conclusion, we have developed an in vitro model for the commencement of ß cell neoplastic transformation. This study indicates that the myc and ras oncogenes are sufficient to initiate transformation. However, considering the addition of TGF{alpha} before or after infection of ß cells, the lack of a difference could be explained by suggesting that an accumulation of genetic events, rather than their order, is important for malignant transformation of cells (40).


    Acknowledgments
 
We are grateful to Dr T.C. Thompson for providing us with the Zipras/myc9 retroviral construct and Dr S. Vuk-Pavlovic for the synthesis of primers. We thank Dr M. Sopta for critical reading of the manuscript. This work was supported by grants 00981103, 00981104 and 00981109 from the Ministry of Science and Technology, Republic of Croatia.


    Notes
 
2 To whom correspondence should be addressed Email: pavelic{at}rudjer.irb.hr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 17, 1998; revised April 1, 1999; accepted May 4, 1999.