Pancreatic adenocarcinomas are frequently associated with an altered synthesis of
mucins (Balague et al, 1994; Hollingsworth et al, 1994; Kim et al, 1999; Andrianifahanana
et al, 2001). Biochemically, mucins are high-molecular-weight glycoproteins and their
polypeptide chains have domains rich in threonine and/or serine, whose hydroxyl groups
are in O-glycosidic linkage with oligosaccharides. These domains are composed of tandemly
repeated sequences that vary in number, length, and amino-acid sequence from one mucin
to another (Gendler and Spicer, 1995; Moniaux et al, 2001). They are produced mainly
by the secretory epithelial cells for the lubrication and protection of ducts and
lumen within the human body (Gendler and Spicer, 1995). In all, 14 human mucin genes
have been identified, designated as MUC1-4, MUC5B, MUC5AC, MUC6-8, MUC11-12, MUC13,
and MUC16-17 (Gendler et al, 1990; Lan et al, 1990; Aubert et al, 1991; Porchet et
al, 1991; Bobek et al, 1993; Dufosse et al, 1993; Gum et al, 1994, 1997; Shankar et
al, 1997; Toribara et al, 1997; Williams et al, 1999, 2001; Yin and Lloyd, 2001; Gum
Jr et al, 2002). Based on the structure, mucins are categorised into three distinct
forms: membrane spanning (MUC1, MUC3, MUC4, MUC12 and MUC17), gel forming (MUC2, MUC5AC,
MUC5B and MUC6), and soluble (MUC7) (Moniaux et al, 2001). MUC4 has been cloned from
the human trachea and human pancreatic tumours, and the full-length cDNA sequence
is known (Nollet et al, 1998; Moniaux et al, 1999; Choudhury et al, 2000a). The NH2-terminus
of MUC4 is composed of a 27-residue signal peptide and a large domain varying in length
from 3285–7285 amino-acid residues as a result of variable number of 16 amino-acid
tandem repeat units (VNTR). The COOH-terminus of MUC4 encodes 12 distinct domains
that include two cysteine-rich domains, three epidermal growth factor (EGF)-like domains,
two regions rich in potential N-glycosylation sites, one hydrophobic transmembrane
region, and one short cytoplasmic tail. Nine out of 12 of the C-terminal domains share
60–80% similarity in sequence with the rat sialomucin complex, known as SMC. Sialomucin
complex is the rat homologue of human MUC4 and is now known as rat Muc4 (Moniaux et
The apomucins MUC1, MUC5B, MUC5AC, and MUC6 are expressed in the normal pancreas;
MUC1, MUC2, and MUC4 are upregulated in pancreatic tumours (Andrianifahanana et al,
2001), whereas MUC5B, MUC5AC, and MUC6 are slightly downregulated. The MUC4 mucin
is expressed at high levels in human pancreatic tumours and tumour cell lines, with
an undetectable level in the normal pancreas (Andrianifahanana et al, 2001). MUC4
is also expressed by metaplastic ducts and its expression increases with higher grade
in pancreatic intraepithelial neoplasias (PanINs) (Swart et al, 2002). The human tissues
showing an undetectable level of MUC4 expression are the gall bladder biliary epithelial
cells, intrahepatic bile ducts, and the liver (Vandenhaute et al, 1997). In contrast,
the MUC4 apomucin is expressed in numerous normal human tissues like the stomach,
ovary, salivary gland, colon, lung, trachea, uterus, and prostate (Audie et al, 1993;
Reid and Harris, 1998; Buisine et al, 1999; Gipson et al, 1999). During foetal development,
there is a complex spatiotemporal regulation of the MUC4 gene in the gastroduodenal
tract and accessory digestive glands.
The membrane-associated mucins rat Muc4 and MUC1 have been reported to play a role
in tumour progression and metastasis (Gendler and Spicer, 1995; Komatsu et al, 1997,
2000; Kim et al, 1999). The implantation of human tumour cells in the pancreas of
nude mice (orthotopic (OT) implantation) has proved to be useful in studying the progression
of pancreatic cancer in vivo (Marincola et al, 1989). The organ microenvironment has
been shown to influence the physiological properties of the tumour cells in the production
of degradative enzymes and the regulation of mdr1 mRNA and P-glycoprotein expression
(Fidler et al, 1994). The survival and growth of a particular tumour cell are significantly
affected by the local milieu provided by a particular organ environment (Fidler et
In the present study, the influence of the local host microenvironment on the expression
of the MUC4 transcript and protein was examined for the first time. Using human pancreatic
adenocarcinoma cell lines, CD18/HPAF and SW1990, human pancreatic tumour xenografts
were developed at the OT and subcutaneous (SC) sites of the nude mouse. The morphologically
differentiated invasive OT tumours demonstrated a high level of expression of MUC4
mRNA and protein compared to undetectable levels in poorly differentiated SC tumours.
However, the in vitro culture of SC tumour cells resulted in the expression of MUC4
transcripts comparable with its expression level in the parental cell line CD18/HPAF.
Paracrine stimulation by growth factors and cytokines has been demonstrated to be
one of the mechanisms responsible for the organ preference and proliferation of the
tumour cells. The MUC4-expressing OT tumours also showed transforming growth factor
(TGF)β2 expression. The study suggests that the site of pancreatic tumour growth in
vivo strongly influences MUC4 and TGFβ2 expression, tumour morphology, and invasiveness
of CD18/HPAF cells.
MATERIALS AND METHODS
Female athymic mice (nu/nu) (6–8 weeks) were obtained from Charles River (Wilmington,
MA, USA). The mice were housed in laminar flow cabinets under specific pathogen-free
conditions. The University of Nebraska Medical Center Institutional Animal Care and
Use Committee approved animal protocols used in this study (IACUC #97-069-03), which
comply with the Public Health Service Policy on the Humane Care and Use of Laboratory
Tumour cell line and tumour cell culture
The CD18/HPAF cell line used in the study was originally derived from the parental
heterogeneous HPAF pancreatic tumour cell line by a limiting dilution technique (Metzgar
et al, 1982; Kim et al, 1989). Cells were cultured in Dulbecco's modified Eagle medium
(DMEM) containing 10% foetal bovine serum (FBS) and penicillin–streptomycin 200 U ml−1
(Life Technologies Inc., Grand Island, NY, USA). The SW1990 cell line was established
from spleen metastasis of grade II pancreatic adenocarcinoma derived from the exocrine
pancreas (Kyriazis et al, 1983). Cells were culture in leibovitz's L-15 medium with
10% FBS and penicillin–streptomycin 200 U ml−1 (Life Technologies Inc.) at 37°C without
CO2. For in vivo injections, cells were harvested from subconfluent cultures by treatment
with 0.05% trypsin and 0.53 mM EDTA (trypsin-EDTA solution; Life Technologies Inc.)
and resuspended in Hank's balanced salt solution (HBSS) for injection. Only single-cell
suspensions with >90% viability were used for injection.
A portion of tumour tissue, obtained 2 weeks after implantation of the CD18/HPAF cells
into the pancreas or the SC tissue of the nude mice, was placed in a 10% DMEM medium
and minced finely with a scalpel. The medium containing the tissue pieces was centrifuged
and the supernatant containing the floating fat tissue was removed. The tissue pellet
was treated with DMEM supplemented with collagenase P (3.75 mg ml−1 medium) at 37°C
for 15 min. The digestion of the tissue was terminated by adding 10% DMEM. After washing
the tissue three times in DMEM medium, tissue fragments were seeded into six-well
plates and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. After 24 h,
tumour cells began to migrate out from the tissue pieces into the surrounding areas.
The wells became subconfluent at day 5 and were trypsinised with Trypsin-EDTA solution
twice for different time periods: first for 1 min to detach and remove the fibroblasts
and second for 5 min to harvest the tumour cells. Cells were washed and seeded in
flasks containing 10% DMEM medium.
OT and ectopic implantation of tumour cells
Tumour cells were harvested from the culture flasks by trypsinisation in EDTA solution,
and were washed by centrifugation in a serum-containing medium. After being washed
twice in PBS pH 7.4 (Life Technologies Inc.), they were resuspended in the same buffer
at a concentration of 10 × 106 cells ml−1. Mice were anaesthetised with 100–200 mg kg−1
ketamine and 5–16 mg kg−1 of xylazine. A volume of 50 μl of cell suspension (10 ×
106 cells ml−1) was injected into different tissues like the pancreas, submandibular
gland (SMG), ovary, stomach, and SC site. After the injection, the organ was returned
to the correct position and the abdomen was sutured using chromic catgut. The skin
was closed with metal clips, which were removed 10 days later. The SC injection was
performed using 50 μl of cell suspension at a site on the back between the scapulae.
After implantation, mice were inspected twice a week. Tumour formation was checked
twice a week in the first 2 weeks and daily thereafter. Tumour-bearing mice were killed
when an intra-abdominal mass measuring ∼2 cm in diameter was palpated. To assess the
tumour dissemination pattern, in each group at least four mice were kept alive until
they were moribund. After sacrifice, primary tumours and the metastatic tumours were
weighed, measured, and cut into small fragments. These fragments were processed for
immunohistochemistry (IHC) or RNA isolation, or were kept in tissue culture medium
and processed to obtain a cell line, as described in the previous section.
Isolation of RNA and Northern blotting
The total cellular RNA from the normal human pancreas, human pancreatic xenografts,
and CD18/HPAF cell line was isolated by the guanidine isothiocyanate and cesium chloride
(CsCl) cushion ultracentrifugation method (Chirgwin et al, 1979). The Northern blots
were performed as described previously (Choudhury et al, 2000b).
Xenographic tumour tissues were fixed in 10% buffered formalin and embedded in paraffin.
Tumour sections (5 μm) were assayed for MUC4 apomucin by using a modification of the
previously described ABC immunohistochemical method (Pour et al, 1993; Batra et al,
1995). Briefly, tissue sections were deparaffinised in xylene, rehydrated in graded
ethanol, and treated for 20 min with 0.3% H2O2/methanol to block endogenous peroxidase.
The sections were blocked with normal goat serum for 1 h, followed by incubation at
4°C overnight with either anti-MUC4 rabbit antiserum raised against 16 amino-acid
tandem repeat peptide (Ser Thr Gly Asp Thr Thr Pro Leu Pro Val Thr Asp Thr Ser Ser
Val) or pre-immune rabbit serum as a negative control. The specificity of the antisera
generated against the tandem-repeat peptide in staining pancreatic tumour cells was
evaluated as described earlier (Choudhury et al, 2000b).
Semiquantitative reverse transcription–polymerase chain reaction (RT–PCR)
Total RNA (0.5 μg) from the tumour tissue or cell lines was reverse transcribed using
the first-strand cDNA synthesis kit (Perkin-Elmer, Branchburg, NJ, USA) and oligo
d(T) primers, according to the manufacturer's instructions. Oligonucleotide primers
to the nontandem repeat region of MUCs 1, 2, 3, 4, 5AC, 5B, 6, 7, TGFβ1, and TGFβ2
are designed from the published sequences in the GenBank, as described earlier (Choudhury
et al, 2000b). The mucin genes and TGFβ were coamplified with the same GAPDH primers.
Amplifications were performed in a programmable thermal controller (PTC-100, MJ Research,
Inc., Watertown, MA, USA). PCR amplification reactions were described previously (Andrianifahanana
et al, 2001; Choudhury et al, 2000b). For convenience, the corrected densitometric
scores for different products were categorised in three different ranges: high value
(+++), moderate value (++), and weak value (+). Each value was determined as the mean
of four densitometry readings.
In vivo tumorigenicity and metastatic behaviour of CD18/HPAF cells
CD18/HPAF cells were implanted orthotopically (OT) or ectopically (SC) in nude mice.
After 7 days, at any given time point, the extent of tumour growth was higher at OT
compared to SC sites. At 20 days after injection, the OT tumour volume was found to
be 2.5-fold higher as compared to the SC tumours, reflected in the tumour weight,
as shown in Table 1
Tumorigenicity and production of spontaneous metastases in CD18/HPAF cells
10 × 106 viable tumour cells were injected into the pancreas or subcutis of two groups
of 10 mice/group.
Days post-injection (±SE), when the tumours could be palpated.
Tumour volume (mm3±s.e.) on day 20 Tumour size was measured with a caliper.
Average of the weight (in grams±s.e.) of the tumours isolated from different mice.
Number of mice with tumours/number of injected mice.
Lymph node (LN) metastases include mediastinal lymph node, mesenteric lymph node,
iliac lymph node, and inguinal lymph node metastases.
. Among the six mice bearing OT tumours, two showed extensive invasion of the stomach
and duodenum, and three showed regional invasion of the stomach and duodenum. Four
mice from each group (OT and SC) were killed after 30 days when they became moribund,
and were dissected to examine the sites of metastasis. Tumours of CD18/HPAF cells
in pancreas (OT tumours) showed a high incidence of metastases to regional lymph nodes
(LNs) and distant metastasis to mediastinal LNs and mesenteric LNs. In contrast, the
SC tumours were confined to the site of injection and none of the mice harbouring
these tumours showed detectable signs of metastases (Table 1). None of these tumours
(OT or SC) showed any signs of necrosis.
Similar results were obtained using another pancreatic tumour cell line, SW1990, where
a significant difference in tumour volume (P<0.01) was observed for the OT tumour
6182+1003 mm3 compared to SC tumour 1774+844 mm3. The OT tumours metastasised to LNs
in all animals compared to no metastasis in the SC tumours.
Expression of MUC4 mRNA by OT and SC tumours
We further analysed the status of MUC4 transcripts in the tumours that are generated
in two different host environments. Total RNA isolated from the tumour cell line (CD18/HPAF),
tumour tissues, and normal human pancreas was fractionated on agarose gel electrophoresis,
Northern blotted, and probed with a MUC4 tandem repeat cDNA probe. As reported in
our previous study (Choudhury et al, 2000b), the MUC4 cDNA probe hybridised to a large-sized
transcript (∼26.5 kb) in CD18/HPAF cells, and showed a smear ranging from 10 to 29 kb
in the OT tumours on Northern blot (Figure 1
Northern blot of total cellular RNA (20 μg) separated in a 1% agarose/formaldehyde
gel from the normal human pancreas tissues, CD18/HPAF cultured cells, and the CD18/HPAF
cells grown as SC and OT tumours. (A) Blot was probed with a 32P-labelled MUC4 tandem
repeat cDNA probe, and the same membrane was stripped and hybridised with a GAPDH
cDNA probe. (B) Densitometric values (±s.e.) for the bands above in three different
experiments were determined by using Molecular Dynamics ImageQuant software program.
Values obtained for the MUC4 smear were divided by the densitometic values for the
). The conceptual expected transcript size of the MUC4 in HPAF cells will be ∼26.5 kb
(Choudhury et al, 2000a). OT tumours showed an MUC4 transcript similar or little higher
than the parental cell line CD18/HPAF (Figure 1). On the other hand, MUC4 was not
detected in the SC tumour, and the value showed in Figure 1 is the background. The
MUC4 mRNA expression in normal human pancreas was below the background level.
Histology and MUC4 protein expression in tumours
We studied the tumour histology of the MUC4-expressing OT as well as the tumours showing
undetectable MUC4 expression, and were interested to see if there is any correlation
with the tumour morphology. Histopathological examination of the tumour tissues stained
with haematoxylin and eosin revealed well-developed duct formation and cellular polarisation
in the OT tumour (Figure 2A
Tumour of CD18/HPAF cells grown in nude mice. (A) OT tumour showing a moderately differentiated
tumour with glandular structures filled with mucin. (B) The same cells grown in SC
tissue, showing an amorphous mass of tumour cells with no signs of differentiation.
Original × 32.
). In contrast, the SC tumour sections showed an amorphous mass of cells with very
little development of ducts in the tumours, and the cells were anaplastic. The tumour
cells lacked cellular polarisation and, therefore, did not form luminal spaces (Figure
The MUC4 protein expressions in OT and SC tumour sections were determined by IHC,
using a rabbit polyclonal antiserum raised against MUC4 (Choudhury et al, 2000b).
The immunoreactivity of the anti-MUC4 antibody was seen in OT tumour sections (Figure
Immunohistochemical staining of CD18/HPAF cells grown in the pancreas of the nude
mouse (A) and in SC tissues (B). The CD18/HPAF tumours in the pancreas show immunoreactivity
to anti-MUC4 antiserum (1 : 100 dilution (A)), whereas the pancreatic tissue of the
nude mouse remains unstained, as do the tumours grown in SC tissue (B). Original ×
50 (A, B).
), but not in SC tumour sections (Figure 3B). The positive staining in the OT tumour
section was specifically blocked by pre-incubation of the MUC4-antiserum with the
tandem repeat peptide (data not shown). The control sera (i.e., the pre-immune sera)
did not show reactivity with any tumours. The OT tumours showed metastases to the
LNs (Table 1). The metastasised LN tumours showed morphology and MUC4 staining similar
to that observed in the OT tumours (data not shown).
In vitro expression of MUC4 mRNA by OT and SC tumour cells
To answer the question if there was a clonal expansion of non-MUC4-expressing cells
in the SC tumours (showing undetectable levels of MUC4), we isolated and cultured
cells from the SC tumours, and studied the MUC4 expression. In SC tumour cells cultured
in vitro, MUC4 mRNA expression appeared gradually and increased from passage 2 to
6, with an expression level similar to MUC4 in the CD18/HPAF parental cell line in
later passages (Figure 4
(A) Northern blot of total cellular RNA (20 μg) extracted from SC tumour cells, OT
tumour cells at different passages and CD18/HPAF cell line, separated in a 1% agarose/formaldehyde
gel. (a) Probed with 32P-labelled MUC4 tandem repeat cDNA probe. (b) The same membrane
as shown in (a), probed with a GAPDH cDNA probe. (B) Densitometric values (±s.e.)
for the bands in three different experiments were determined by using Molecular Dynamics
ImageQuant software program. Values obtained for the MUC4 smear were divided by the
densitometric values for the GAPDH band.
). OT tumour cells in culture showed a transient decrease in the level of MUC4 transcripts
and also exhibited a level comparable to MUC4 in CD18/HPAF (Figure 4) in the later
TGFβ expression in tumour cells, OT and SC tumours
Previously, we demonstrated a positive correlation in the expression of MUC4 and TGFβ2
transcripts (Choudhury et al, 2000b). For this analysis, the expression of TGFβ1 and
TGFβ2 was studied by RT–PCR using total RNA isolated from CD18/HPAF cells and OT and
SC tumours (Figure 5
(A) Analysis of TGFβ1 and TGFβ2 expression in CD18/HPAF cells, OT tumours, and SC
tumours. Total RNA was isolated; TGFβ and GAPDH mRNA are coamplified in each reaction
by RT–PCR. (B) The band intensity of the amplified products was quantified for each
sample using the gel expert™ 3.5 software suite. The densitometric values (±s.e.)
for the bands in three different experiments were calculated for a gene-specific product
and GAPDH for each reaction. The value for a gene-specific product is expressed per
unit of GAPDH to account for any differences in the starting amounts of RNA. OT, orthotopic
tumour; SC, subcutaneous tumour; S, serum; SF, serum-free.
). We found that OT tumours showed TGFβ2 expression with a range two-fold higher than
the parental cell lines CD18/HPAF. However, the SC tumour samples showed no undetectable
TGFβ2 expression. Sometimes, a very low level of TGFβ2 was detected in SC tumours
(two out of the six) without expression of MUC4 (data not shown). On the other hand,
the expression of TGFβ1 was found to be similar in tumours (OT and SC) and the CD18/HPAF
As a control, another cell line, SW1990, was used to validate the results. The SW1990
line was implanted in OT and SC sites in nude mice. Expression of MUC4 as well as
TGFβ2 was investigated. The SW1990 OT exhibited a high level of MUC4 and TGFβ2, as
compared to the SW1990 parental line or the SW1990 SC (data not shown).
Expression of mucin genes in normal human pancreas, CD18/HPAF cell line, OT and SC
To obtain a comparative picture of MUC4 expression along with other mucin genes, RT–PCR
amplification was performed using mucin gene-specific and GAPDH primers designed from
the published sequences in the GenBank. A comparison of mucin gene expression in the
normal human pancreas tissue, pancreatic tumour cell line (CD18/HPAF), and tumour
tissues (OT and SC) is shown in Table 2
RT–PCR expression analysis of mucin genes expression
Three orthotopic (OT), three subcutaneous (SC), and seven normal human pancreas were
analysed. +++, high; ++, moderate; +, low; −, undetectable; ND, not determined.
. Consistent with the Northern blot and IHC, RT–PCR showed no expression of MUC4 in
the normal human pancreas and the SC tumours, whereas a high level of MUC4 expression
was found in the CD18/HPAF cell line and the OT tumours. The expression of MUC1 and
MUC5B appeared similar in all the samples. MUC2 was detected only in the tumour cell
line, but not in the normal human pancreas and tumour samples. The expression of MUC5AC
was weak in OT and SC tumours, with traces in the cell line and normal human pancreas.
MUC6 was detected at a high level only in the normal pancreas. MUC3 and MUC7 mRNA
expression was not detected in the CD18/HPAF cell line and tumour samples. The positive
controls (as mentioned in the Materials and methods section) for MUC2, MUC3, MUC5AC,
MUC5B, and MUC7 showed mucin expression. Among eight mucin genes analysed, MUC4 was
the only gene that showed high levels of expression in OT tumours, with no detectable
expression in the normal pancreas or SC tumours. For PCR analysis, primers were designed
in the non-tandem repeat regions of the human mucin genes. The amplified PCR products
for each mucin gene showed the expected size with 100% sequence identity to the corresponding
human sequences, thereby ruling out the possibility of amplification of the mouse
The MUC4 mucin exhibits a pancreatic tumour-associated expression, with no detectable
expression in a normal pancreas (Balague et al, 1994; Hollingsworth et al, 1994; Kim
et al, 1999; Choudhury et al, 2000a; Andrianifahanana et al, 2001). Based upon the
structural information, MUC4 has been proposed to exist as a heterodimeric molecule
consisting of a large mucin-type subunit and a membrane-anchored subunit with three
EGF-like domains (Moniaux et al, 1999; Choudhury et al, 2000a). It has been proposed
that the MUC4 mucin may have growth factor-like properties, because its rat homologue
(rat Muc4) is referred to interact with the oncogene p185neu (Carraway et al, 1999).
Overexpression of SMC (rat Muc4) has been shown to promote tumour growth in primary
tumours and has resulted in metastasis (Komatsu et al, 2000).
In the present study, the effect of the host local environment on MUC4 expression
was examined in nude mice. The clonal human pancreatic cancer cell line CD18/HPAF
that expresses a high level of MUC4 mRNA (Choudhury et al, 2000b) was used for generating
human pancreatic xenografts at different sites in nude mice. We observed significantly
higher tumour growth (P<0.01) rates after implantation at OT sites compared to SC
sites in nude mice. In addition, the OT tumours also showed a high incidence of metastasis
to regional LNs, and distant metastasis to mediastinal LNs and mesenteric LNs. A high
level of MUC4 transcripts and proteins was observed in the human pancreatic tumour
xenografts at an OT site, a site analogous to human pancreas that does not show MUC4
expression, compared to SC sites. When tumours were generated at other MUC4-expressing
sites in nude mice like SMG and stomach, these tumours also showed equivalent levels
of MUC4 (unpublished result). Pancreas, SMG, and stomach, being physiologically active
organs that are well perfused compared to the SC environment, have spare vasculature.
The MUC4-expressing cells, when grown in the well-vascularised site (OT), revealed
a high level of MUC4 expression, as compared to a less-vascularised (SC) environment.
The observation suggests a role of serum factors in regulating the MUC4 expression,
or there may be a clonal expansion of a non-MUC4-expressing cell type in the SC tumours.
To answer these questions, we harvested SC tumour cells and cultured them in vitro.
The in vitro culture of SC tumour cells returned the expression of MUC4 transcripts
to the parental cell line level, further suggesting a role of serum factor(s) in regulating
MUC4 expression. Our earlier study also demonstrates a serum-dependent increase in
MUC4 expression in human pancreatic tumour cells (Choudhury et al, 2000b).
Further, histological examination of the tumours revealed the OT tumours as moderately
differentiated, whereas the SC tumours were poorly differentiated, suggesting that
the expression of MUC4 could be influenced by the differentiation grade of tumours.
We have made similar observations on a panel of pancreatic tumour cell lines, where
a majority of differentiated adenocarcinomas showed higher levels of MUC4 transcripts
compared to cell lines derived from poorly differentiated adenocarcinomas (Hollingsworth
et al, 1994; Choudhury et al, 2000a; Andrianifahanana et al, 2001).
The lack of detectable expression of MUC4 in SC tumours could also be due to paracrine
regulation from the surrounding tissue environment that may be blocking the transcription
of MUC4. Paracrine stimulation by growth factors and cytokines has been demonstrated
to be one of the mechanisms responsible for the organ preference and proliferation
of the tumour cells. In the human colon carcinoma cell line, paracrine stimulation
by a soluble factor from human colon connective tissue was involved in inducing the
expression of the MUC1 mucin in vitro (Irimura et al, 1990). Cytokine-like tumour
necrosis factor-α, interleukins, and EGF have been shown to be involved in the regulation
of mucin gene expression (Dabbagh et al, 1999; Longphre et al, 1999; Takeyama et al,
1999; Kim et al, 2000; Smirnova et al, 2000). One of the cytokines (i.e. TGF-β) showed
an increased expression in many advanced human cancers (Gorsch et al, 1992; Gold et
al, 1994) including pancreatic cancer (Friess et al, 1993a, 1993b). A TGFβ2-dependent
increase in MUC4 expression in pancreatic adenocarcinoma (Choudhury et al, 2000b)
and elevated levels of TGFβ2 transcripts in MUC4-expressing OT tumours suggest the
involvement of this cytokine in MUC4 regulation by autocrine and/or paracrine manner
in CD18/HPAF tumours. Nevertheless, the expression of MUC4 is also regulated by TGFβ2,
post-translationally and post-transcriptionally in normal and mammary adenocarcinoma
cells, respectively (Price-Schiavi et al, 1998, 2000).
Earlier studies have shown that the organ environment can influence tumorigenesis;
production of degradative enzymes; melanin and angiogenic molecules; induction of
terminal differentiation; level of P-glycoprotein associated with the multiple drug-resistance
phenotype; and IL-8 expression (Hart and Fidler, 1980; Price et al, 1988; Nakajima
et al, 1990; Staroselsky et al, 1990; Fabra et al, 1992; Wilmanns et al, 1992; Radinsky
et al, 1994; Gutman et al, 1995). The influence of the organ environment on the growth
of tumour cells was originally proposed in Paget's hypothesis of the ‘seed and the
soil’ (Paget, 1989).
In summary, we believe that this is the first study showing in vivo regulation of
human MUC4 expression in pancreatic tumours. The expression of MUC4 was high in moderately
differentiated tumours, with undetectable levels in poorly differentiated SC tumours.
The OT tumours also showed metastases not only to the regional but also to the distant
LNs. The SC tumour cells, when cultured in vitro, showed MUC4 expression, suggesting
a role of serum factor(s) in its regulation. Our results also indicated a direct correlation
between the MUC4 expression and the levels of TGFβ2 transcripts in the CD18/HPAF tumours,
as well as in CD18/HPAF cells in vitro, as described earlier (Choudhury et al, 2000b).
These results reveal that the local host environment regulates the MUC4 expression
in pancreatic tumours (CD18/HPAF) under in vivo conditions.