0021-972x196/$03.00/0
Journal
of Clinical
Endocrinology
and Metabolism
Copyright
0 1996 by The Endocrine
Society
Vol. 81, No. 3
Printed
in U.S.A.
Hypercalcemia
Associated
with Infantile
Producing
Parathyroid
Hormone-Related
TOSHIMI
MICHIGAMI,
HIDEYUKI
YAMATO,
MASAHIRO
NAKAYAMA,
AKIHIRO
YONEDA,
KENJI
IMURA,
AND KEIICHI
OZONO
SOTARO
KENICHI
Fibrosarcoma
Protein
MUSHIAKE,
SATOMURA,
Departments
of Pediatrics,
Pediatric Surgery, Clinical Laboratory,
and Environmental
Medicine,
Osaka
Medical Center and Research Institute for Maternal
and Child Health, 840 Murodo-cho,
Izumi,
Osaka 590-02;
and Kureha Chemical Industry
Co., 3-26-2 Hyakunin-cho,
Shinjuku-ku,
Tokyo 169,
Japan
ABSTRACT
We describe
a 7-month-old
boy who manifested
severe hypercalcemia associated
with mesenchymal
neoplasm.
A huge hypervascular
tumor
on the neck had been detected
in prenatal
ultrasonography.
Surgical
removal
of the entire
tumor
at birth
was not indicated,
because the tumor
was diagnosed
as hemangioma.
Chemotherapy
and radiotherapy
were attempted,
but there was no effect on tumor
growth.
When the infant was 6 months
old, the serum calcium
level
increased
rapidly,
associated
with the expansion
of the tumor.
Hypophosphatemia
due to phosphaturia
was also observed.
Serum PTH
was undetectable,
whereas
the serum concentration
of carboxyl-terminal (C-terminal)
fragments
of PTH-related
protein
(PTH-rP)
was
markedly
elevated.
Northern
blot analysis
and immunostaining
demonstrated
the expression
of PTH-rP
in the tumor.
The tumor
was
transplantable
to nude mice and caused
elevation
of circulating
PTH-rP
in the animals.
Histological
examination
ofthe patient’s
bone
revealed
an increased
number
of osteoclasts.
These findings
were
consistent
with humoral
hypercalcemia
of malignancy
caused by the
excess production
of PTH-rP.
The tumor was identified
histologically
as infantile
fibrosarcoma,
which has not been reported
as a cause of
humoral
hypercalcemia
of malignancy
to date. The expression
of
PTIGPTH-rP
receptor
messenger
ribonucleic
acid was detected in the
tumor by the RT-PCR,
suggesting
that PTH-rP
may have exerted its
effect in the tumor in an autocrine/paracrine
manner.
In addition
to
the systemic
effect of PTH-rP
manifested
as hypercalcemia,
the
PTH-;P
secreted
from the neoplasm
could have been a local factor
involved
in the growth
of the tumor.
(J Clin Endocrinol Metab 81:
1090-1095,
1996)
I?
tumors. For example, a transcription factor, Tax, is reported
to stimulate the transcription of the PTH-rP gene in adult T
cell leukemia (lo), and the site-specific demethylation of the
promoter region in the PTH-rP gene appears to be fundamental in controlling the gene expression in renal carcinoma
cell lines (11).
PTH-rP exerts its function through an effector system almost identical to that of PTH, involving an intramembranous
receptor whose complementary DNA (cDNA) has been
cloned recently, stimulating the accumulation of both CAMP
and inositol triphosphates (12-14). In addition, CAMP is reported to be one of the transduction signals that induces the
activity of the PTH-rP gene promoter (15, 16). Therefore, it
is reasonable to postulate that PTH-rP itself stimulates the
production of the hormone when the cells producing PTH-rP
express the PTH/PTH-rP receptor.
As noted above, PTH-rP is expressed in normal tissuesas
well as in malignancies, and accumulating evidence shows
that PTH-rP exerts various physiological functions. One of
these functions, the stimulation of growth by PTH-rP, has
been reported in many kinds of cells (17-19). In this sense,
PTH-rP can act as a local growth factor. HHM is the most
prominent sign of the calcium-regulating effect of PTH-rP,
but hypercalcemia is thought to be elicited after the serum
concentration of the hormone is elevated to levels higher
than normal. Based on findings that show PTH-rP to be a
local growth factor, we suggest that PTH-rP may promote
tumor growth in an autocrine/paracrine manner, resulting
in excessproduction and the acceleration of HHM.
protein (PTH-rP) was initially identified
as a causal factor of humoral hypercalcemia of malignancy (HHM) (l-3). HHM occurs in association with a variety of malignant tumors, including squamous cell carcinoma
of the lung, renal cell carcinoma, breast cancer, and
adult T cell leukemia (4, 5). PTH-rP is also expressed in
TH-RELATED
normal
tissues,
such
as the placenta
and
lactating
mammary
gland, and in the fetus in many sites; however, its physiological functions are not fully understood (6,7). Knocking out
the PTH-rP gene causesa chondrodysplasia-like phenotype,
which suggests that the peptide plays an essential role in
skeletal development (8). The malignancies causing HHM
frequently originate in cellsand tissuessuch askeratinocytes,
islet cells, mammary glands, and kidneys, which secrete
PTH-rP even in the normal state. Therefore, failure to control
PTH-rP gene expression in the courseof tumorigenesis could
lead to the excessproduction of PTH-rP in malignancies. In
this sense,not the ectopic but the eutopic excessproduction
of PTH-rP is thought to be a cause of HHM. Why certain
neoplasmscome to produce excessPTH-rP is still unknown,
but malignant transformation seemsto play a key role in the
phenomenon (9). The molecular mechanism responsible for
the excessproduction of PTH-rP hasbeen reported in certain
Received
May 1, 1995. Revision
received
September
12, 1995. Accepted September
18, 1995.
Address
all correspondence
and requests for reprints
to: Dr. Keiichi
Ozono, Department
of Environmental
Medicine,
Osaka Medical Center
and Research Institute for Maternal
and Child Health, 840 Murodo-cho,
Izumi, Osaka 590-02,
Japan.
1090
PTH-rP PRODUCTION
HHM is one of the most common paraneoplastic syndromes in adults. In contrast, pediatric HHM is relatively
rare (20). The reason for this is, in part, the difference in the
variety of malignant tumors shown in adults and children.
Despite the low frequency of HHM in children, the pediatrician who treats patients with malignancies should be
aware of this condition, because hypercalcemia is one of the
adverse, but controllable, factors. Here we describe a patient
with hypercalcemia due to a PTH-rP-producing
tumor. Both
his age and the histological characteristics of the tumor were
rare in terms of patients with hypercalcemia associated with
the excess production of PTH-rP.
Case Report
A 30-yr-old
woman was admitted
to the perinatal
unit of our hospital
in the seventh month of pregnancy
because her baby had been diagnosed
nrenatallv
with a large neck tumor. A bov was born at the 36th week of
gestation:
His birth weight was 2840 g, and Apgar scores at both 1 and
5 min after birth were 8. A huge mass was prominent
on the neck. The
surface appearance
of the neck tumor gave rise to a clinical impression
of hemangioma,
and we assumed
that this would
be impossible
to
remove
surgically,
since the tumor was rich in blood vessels, and the
vena cava was involved
in the mediastinal
portion
of the tumor. Thus,
chemotherapy
combined
with radiation
therapy was selected as the first
treatment.
However,
the administration
of aspirin and cyclophosphamide had little effect, and high dose of methylprednisolone
treatment
also had only limited
effects on the tumor. The tumor came to involve
the mediastinum,
and the mass in the mediastinum
grew more rapidly
than that in the neck portion. When the boy was 6 months old, his serum
calcium
(Ca) concentration
began to increase in association
with the
accelerated
growth
of the tumor.
The growing
tumor obstructed
the
airway,
and the patient required
intubation
and respiratory
support.
When he was 7 months old, he manifested
severe hypercalcemia
(22.6
mg/dL),
although
he did not present obvious
clinical symptoms,
such
as dehydration,
unconsciousness,
or circulatory
problems.
Combination
therapy
consisting
of rehydration
and the administration
of diuretics
and glucocorticoids
was effective,
resulting
in a decrease in the serum
Ca level to 12-13 mg/dL.
Radiographic
examinations
revealed
a reduction in bone mineral density in the limbs and no metastatic
lesions in the
bones. Nephrocalcinosis
was also observed.
His general condition
was
too poor for a radioisotope
bone scan to be performed.
When the child
was 8 months old, surgical reduction
of the tumor
was attempted
to
resolve the respiratory
problem,
but excessive
loss of blood from the
large vessels caused the patient to die during the operation.
Two large
masses of the tumor and several smaller masses were resected. The sizes
of two large masses were 11 X 6 X 3 and 7 X5 X 4 cm, respectively.
The
resected specimen
was elastic and soft generally,
but somewhat
firm in
parts; the cut surface was milk-white,
with focal hemorrhage
and necrosis evident.
The cultured
tumor cells were found to have a chromosome
aberration: 46,XY, t(l;S)(qll;q22)
in 9 cells, 46,XY, t(3;6)(p13;q21)
in 4 cells, and
46,XY in 17 of 30 analyzed
cells.
Measurements
metabolism
Materials
and Methods
of biochemical
markers of calcium
Serum PTH was measured
with a midportion
PTH assay (Yamasa
Shoyu, Chiba, Japan) and an intact PTH assay (Nichols
Institute,
Los
Angeles, CA). Serum C-terminal
fragments
of PTH-rP were determined
by RIA with antihuman
PTH-rP-(109-141)
antibody
(Dai-ichi
Radioisotope, Tokyo, Japan) (21). The phosphorus
concentrations
in serum and
urine were determined
by autoanalyzer
(Synchoron
Clinical
System
CX7, Beckman,
Tokyo). Plasma and urinary
CAMP levels were measured
by RIA (Yamasa Shoyu). Serum vitamin
D metabolites
concentrations
were
determined
by a high performance
liquid
chromatography
method,
as reported
previously
(22).
IN FIBROSARCOMA
Histological
examination
of the tumor
The tumor obtained
at surgical intervention
was examined
histologically.
The formaldehyde-fixed
paraffin-embedded
specimen
was
stained with hematoxylin
and eosin. Immunoreactivity
for vimentin,
myoglobin,
desmin, factor VIII, and cu,-antitrypsin
(Dako Japan, Kyoto,
Japan) was also investigated.
Immunohistochemical
staining
was carried out on frozen
sections,
using the avidin-biotin-peroxidase
technique, employing
polyclonal
antiserum
raised against N-terminal
(I-34)
or C-terminal
(127-141)
fragments
of human PTH-rP as first antibodies
(Mitsubishi
Petrochemical
Co., Tokyo, Japan).
Histological
examination
Tissue from the
The specimen
was
room temperature,
Tartrate-resistant
using a commercial
Northern
of the bone
sternum
was obtained
from the patient at operation.
decalcified
overnight
in 5% formic acid solution
at
sectioned,
and stained with hematoxylin
and eosin.
acid phosphatase
(TRAP)
staining
was performed,
kit (Sigma Diagnostics,
St. Louis, MO).
blot analysis
The expression
of PTH-rP in the tumor was investigated
by Northern
blot analysis. Total ribonucleic
acid (RNA) was prepared
from the tumor
tissue of the present hypercalcemic
patient by a guanidium
thiocyanatephenol method (23). Total RNA extracted
from the rhabdomyosarcoma
tissue obtained
from another child was used as a negative
control. Total
RNA prepared
from squamous
cell carcinoma
of the oral cavity that had
been reported
to produce
PTH-rP was used as a positive
control (24).
Twenty
micrograms
of total RNA from each tumor
were electrophoresed on 1% agarose gel containing
5.4% formaldehyde,
and then transferred onto positively
charged nylon membranes
(Amersham
International, Aylesbury,
UK). The transferred
RNA was hybridized
with fulllength human PTH-rP complementary
DNA (generously
donated by Dr.
T. J. Martin,
Victoria,
Australia)
(1). The probe
was labeled
with
[32P]deoxy-CTP
by the random
primer method, and hybridizations
were
performed
at 45 C overnight
in 50% formamide,
5 X SSPE, 5 X Denhardt’s solution,
0.1% SDS, and 100 pg/mL
salmon sperm DNA (all final
concentrations).
Hybridized
filters were washed twice in 0.1% SDS-O.1
x saline sodium
citrate (SSC) for 30 min at 60 C. The hybrids
were
visualized
by autoradiography.
Transplantation
of the tumor
The tumor was cut into 2-mm pieces, and these were inoculated
under
the dorsal skin of 6- to 7-week-old
male nude mice or SCID mice and
allowed to form masses. Three months after transplantation,
one mouse
of each species was anesthetized
with ether and killed, and the formed
mass in the nude mouse was retransplanted
to nude rats. Blood samples
were obtained,
and the concentration
of intact PTH-rP was determined
using an immunoradiometric
assay with two antibodies,
against PTHrP-(l-34)
and PTH-rP-(50-87),
respectively
(Mitsubishi
Petrochemical
Co.) (25). Pathological
investigations
of the formed
masses were also
performed.
RT-PCR
The expression
of PTH/PTH-rP
receptor
in the tumor was investigated using RT-PCR.
Total RNA (5 wg) was incubated
with 50 ng
random
hexamer
(Life Technologies,
Grand Island, NY) at 70 C for 18
min and chilled on ice, then converted
to single stranded
cDNA bv RT,
using reverse transcriptase
(Life Technologies)
according
to the manufacturer’s
recommendations.
Amplification
of cDNA by the PCR was
performed
using two specific oligonucleotide
primers.
These primers
were svnthesized
based on the human PTH/PTH-rP
cDNA sequence
reported
in a previous
paper (14). The sequences of the primers
were
5’-ACAAGGGATGGACATCTGCGT-3’
and
5’-CACAGCGTCCTTGACGAAGAT-3’.
PCR was carried out for 30 cycles, consisting
of 1 min
at 94 C, 1 min at 55 C, and 1 min at 60 C in a 25-FL volume
of reaction
mixture
containing
5 FL first strand cDNA, reaction buffer [containing,
final concentrations,
10 mmol/L
Tris-HCl
(pH 8.3), 50 mmol/L
KCI, 1.5
mmol/L
MgCl,,
and 0.001% gelatin],
deoxy-NTP
mix (0.2 mmol/L
each), the pair of primers
(10 Fmol/L
each), and 2 U recombinant
Tuq
DNA polymerase
(Takara Shuzo, Shiga, Japan). Using 1 PL of the 20-fold
MICHIGAMI
ET AL,.
JCE & M . 1996
Vol81
. No 3
diluted first PCR product
as template,
another
set of 30 cycles of PCR
was performed
in 25 c;L mixture.
The second PCR products
were separated on 2.5% agarose gels containing
ethidium
bromide
and visualized
under ultraviolet
light. The sizes of the fragments
were confirmed
by
reference to a mol wt marker,
and the PCR products
were characterized
by sequencing.
Biochemical
markers
Results
of calcium metabolism
Blood and urine samples were obtained before specific
treatment was given, when the patient manifested severe
hypercalcemia (serum Ca, 22.6 mg/dL). The biochemical
markers of Ca metabolism were evaluated as described in
Materials rindMethods; the values before the administration of
saline or diuretics are summarized in Table 1. The urinary
Ca/creatinine excretion ratio was 6.5, indicating severe hypercalciuria. The percent tubular phosphorus reabsorption
was reduced, suggesting that hypophosphatemia (2.5 mg/
dL) was caused by phosphaturia. Despite the apparent hypophosphatemia, serum PTH, evaluated by both intact and
midportion assays,was undetectable. In contrast, the serum
level of PTH-rP C-terminal fragments was markedly elevated. Nephrogenous CAMP was not elevated, although it is
difficult to set a normal range in infants. The concentration
of 1,25-dihydroxyvitamin D was low for the infant’s age.
Pathological
examination
of the resected tumor
The proliferating ovoid and spindle cells were densely
packed, and interlacing cords and intersecting fascicles of
cells showed a herring bone-like pattern typical of congenital
infantile fibrosarcoma (26) (Fig. 1A). The mitotic rate was
significantly elevated, and dense karyorrhectic nuclei were
sometimesobserved. Immunohistochemical examination revealed immunoreactive staining for vimentin in tumor cells,
but no staining was observed for myoglobin, desmin, factor
VIII, or cu,-antitrypsin. These findings were consistent with
the histological features of infantile fibrosarcoma (26). Antibodies against C-terminal fragments of human PTH-rPstained tumor cell cytoplasm selectively (Fig. 1B). Immunoreactivity for N-terminal PTH-rP was also detected (data not
shown). The histological findings in the massesformed in
nude mice in which the tumor had been transplanted were
similar to the findings in the original tumor, showing evident
nuclear condensation and mitosis (Fig. 1C).
TABLE
1. Biochemical
markers
of calcium
metabolism
Values
in
patient
Serum Ca (mg/dL)
Serum phosphate
(mg/dL)
Urinary
calcium/creatine
ratio
% TRP (%)
Intact PTH (pg/mL)
HS-PTH
(pg/mL)
PTHrP
C-terminal
(pmol/L)
25-Hydroxyvitamin
D (ng/mL)
1,25-Dihydroxyvitamin
D (pg/mW
24.25-Dihvdroxwitamin
D (ne/mL)
ND,
tion.
Not
detected.
% TRP,
Percent
22.6
2.5
6.5
64
ND
ND
900
13.6
10.6
ND
tubular
phosphorus
Normal
range
9.0-11.0
2.7-4.4
co.2
X5
lo-65
MO-560
13.8-55.3
10-29
33-118
0.6-2.6
reabsorp-
FIG. 1. A, Hematoxylinand eosin-stained
section of the tumor
resected from the patient.
The proliferating
ovoid and spindle cells are
densely
packed,
and interlacing
cords and intersecting
fascicles of
cells show a herring
bone-like
pattern
typical
of congenital
infantile
fibrosarcoma.
B, Immunohistochemical
examination
of the tumor using the antibody
against
C-terminal
fragment
of human
PTH-rP.
PTH-rP
immunoreactivity
is present
in the cytoplasm
of the tumor
cells selectively
(arrow).
C, Hematoxylin
and eosin staining
of the
mass formed
in a nude mouse in which the tumor
had been transplanted.
The histological
findings
were very similar
to the findings
in the original
tumor,
showing
evident
nuclear
condensation
and
mitosis.
PTH-rP PRODUCTION
Pathological
of the bone
examination
A large number of tartrate-resistant acid phosphatase-positive cells that were supposed to be osteoclastswere present
in the bone specimen (data not shown).
IN FIBROSAFXOMA
1093
TABLE 2. Effect of the tumor resectionon serumCa and
PTH-rP concentrationsin tumor-bearingnuderats
Serum.ca
Day 0
Synthesis
of PTH-rP
in tumor
cells
Northern blot analysis of total RNA prepared from the
tumor of the hypercalcemic patient, using PTH-rP cDNA as
probe, revealed one major hybrid signal, whose size was 1.5
kilobases. Squamous cell carcinoma, which was used as a
positive control, contained the multiple transcripts for
PTH-rP at 1.5 and 2.1 kilobases,as previously reported (24).
On the other hand, total RNA extracted from the rhabdomyosarcoma did not hybridize with PTH-rP cDNA (Fig. 2).
Transplantation
of the tumor
The transplantation experiment revealed that the tumor
was transplantable to both nude and SCID mice. The formed
masseswere histologically similar to the original tumor, as
noted above. No metastatic tumor was observed. The circulating intact PTH-rP concentration was elevated in the tumor-bearing mice compared with that in the control mice
(2.8-3.5 pmol/L us. undetectable levels). Tumor growth was
accelerated in nude rats, resulting in the elevation of serum
PTH-rP and severe hypercalcemia within 8-9 weeks in those
rats (Table 2). Resection of the tumor was performed in two
of those rats, and serum Ca levels were restored 3 days after
resection in both (Table 2).
23
1
Serum PTH-rP
(pmol/L)
(mg/dL)
Tumor-bearingrats
No. 1
No. 2
Day 3
19.1
11.3
15.5
10.9
Control rats (n = 3)
10.7 * 0.1
Day 0, The day of resection.
Expression
of PTHIPTH-rP
Day 0
Day 7
13.4
2.8
6.7
2.1
2.5 t 0.5
receptor
Using RT-PCR, we detected the expression of PTH/
PTH-rP receptor in the tumor (Fig. 3). The specific signal was
visible but very weak, so we carried out a second PCR, as
described in Materials and Methods. The sequenceof the amplified fragments for PTH/PTH-rP receptor cDNA was verified by sequencing (data not shown).
Discussion
We reported an infantile patient with HHM. The serum
level of C-terminal PTH-rP was markedly elevated. Expression of PTH-rP in the neoplasm at both the protein and
messengerRNA (mRNA) levels was confirmed by immunohistochemistry and Northern blot analysis, respectively.
The tumor was transplantable to both nude and SCID mice
and caused an elevation of circulating PTH-rl?. These findings strongly indicated that the hypercalcemia in the patient
was due to excessPTH-rP produced by the tumor. The tumor
was identified histologically as infantile fibrosarcoma. HHM
is usually causedby carcinoma or hematological malignancy
(4,5), and sarcoma (malignant mesenchymoma) is rare as a
cause of HHM. To our knowledge, this is the first report to
describe an infantile fibrosarcoma that caused humoral hypercalcemia. Interestingly, infantile fibrosarcoma is a controversial lesion that represents an indistinct entity between
the fibromatoses and adult-type fibrosarcoma (26). The histological findings of infantile fibrosarcoma are similar to
1234
-
f
f
28s)
18s
532 bps
FIG. 3. Detectionof PTWPTH-rP receptormRNA in infantile fibro-
+
FIG. 2. Detection of PTH-rP mRNA in infantile fibrosarcomaby
Northern blot hybridization. As a positive control, squamouscell
carcinomaof the oral cavity, which had beenrepoi%edto produce
PTH-rP,wasused(lane1)(24).Twentymicrograms
eachoftotal RNA
from rhabdomyosarcoma
(lane2) andinfantile fibrosarcoma(lane3)
wereapplied,electrophoresed,
andtransferredto a nylon membrane.
Thefilters werehybridizedwith the completecDNAof F’TH-rP(upper
panel). The 28s and 18s ribosomalRNAs visualizedby ethidium
bromidestainingof the gel are shownbelow each lane.
sarcomaby RT-PCR.Total RNA (5 pg eachsample)wasconvertedto
singlestrandedcDNA.PCRwascarriedout asdescribedin Materials
and Methods,
usingspecificprimerssynthesizedfor the PTH/PTH-rP
receptor. The PCR productswere analyzedby ethidium bromide
stainingofthe gel.Lane1,Molwt.marker(~X174MincII
digest); lane
2, human osteoblastic cell line, Saos-2, previously shown to express
the PTHiPTHrP receptor (14); lane 3, infantile fibrosarcoma. The
arrow indicates the position of the specifically amplified fragments of
532 bp. Note that the first and second PCR products were applied in
lanes 2 and 3, respectively. As a negative control, the same RT-PCR
procedure was carried out without RNA, and no specific fragment was
amplified after 2 sets of 30 cycles of PCR (lane 4). The level of expression of the PTIWPTH-rP receptor in infantile fibrosarcoma was
less than that in Saos-2 cells.
1094
MICHIGAMI
those of fibrosarcoma
occurring in adults. However, infantile
fibrosarcoma
is associated with clinical characteristics
that
differ markedly from those of adult fibrosarcoma;
in infantile
fibrosarcoma,
metastasis is extremely rare, and the prognosis
is fairly good with wide local excision (26). In other words,
the clinical course of infantile fibrosarcoma
is benign rather
than malignant.
With the exception of pheochromocytoma,
benign tumors that cause hypercalcemia
are rare (4), and
oncogene activation
is suggested
to play a key role in the
mechanism
by which malignant
cells produce excess PTHrP, although this mechanism
is not clear (9). Taken together,
the features in the present patient indicate the potential malignant properties
of infantile fibrosarcoma.
Infantile
fibrosarcoma
is assumed to be derived from fibroblasts, with no other evidence of cellular differentiation
(27). Fibroblasts
are not reported to produce PTH-rP physiologically,
although
a wide variety of cells do express
PTH-rP in the course of normal fetal development
and in
normal adult tissue (6,7). Therefore, it is not surprising
that
there is no report of humoral hypercalcemia
associated with
fibrosarcoma,
and it is reasonable
to consider that in this
patient, PTH-rP was produced
ectopically
in the tumor.
It has been reported that fibroblasts are a target of PTH-rP,
and this phenomenon
would
suggest that the peptide secreted from the tumor could exert some action on the fibroblastic tumor cells in an autocrine/paracrine
manner (28). As
reported by Kremer et al. (29), human keratinocytes
are another example of target cells in which PTH-rP exerts a paracrine effect. Further, it has been reported
that polyclonal
PTH-rP antiserum
almost totally inhibited
the growth
of a
human renal cell carcinoma cell line (19). The suppression
of
PTH-rP excretion by vitamin D derivatives
is associated with
the reduced growth of MT-2 cells, an adult T cell line (30).
This finding may also support the idea that PTH-rP is an
autocrine/paracrine
growth factor. These findings indicate
that cell proliferation
is regulated
by PTH-rP in a different
manner depending
on the type of cell, and the findings raise
the possibility
that PTH-rP may be involved in the autocrinal
regulation
of growth in some tumors. It is important
to investigate further whether PTH-rP exerts a local effect as well
as a systemic hypercalcemic
effect. As a first step to test
whether this may have occurred in our patient, we analyzed
the expression
of PTH/PTH-rP
receptor in the tumor, although it is doubtful
that all of the effects of PTH-rP are
exerted via the known receptor. We detected the receptor by
RT-PCR, but the expression seemed very weak. The expression of PTH/PTH-rP
receptor in the tumor suggests that the
tumor itself could be the target of the signal transmitted
by
PTH-rP. The small amount of PTH/PTH-rP
receptor mRNA
could have been due to down-regulation
by PTH-rP. These
results support the idea that PTH-rP acted as a growth factor
for the tumor. Alternatively,
a decreased number of PTH/
PTH-rP receptors may alter the sensitivity to the differentiation effect that PTH-rP might have. The successful implantation of this tumor in nude mice and rats allows us to
investigate the effect of PTH-rP on tumor growth both in viva
and in vitro.
In summary,
this patient with HHM associated with infantile fibrosarcoma
prompted
us to reconsider
the relationship between HHM and malignancy,
and the detection of
ET AL.
JCE & M . lYY6
Vol81 . No 3
PTH/PTH-rP
receptor expression
in the tumor led us to
consider the action of PTH-rP on the tumor itself.
Acknowledgments
We thank
Dr. Kyoji
Ikeda for generously
carcinoma
of the oral cavity.
We are grateful
Shigeo
Nakajima
for their useful
discussions.
Yamagata
for her technical
assistance.
providing
us squamous
cell
to Drs. Kanji
Yamaoka
and
We also thank
Ms. Masayo
References
1. Mangin
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
M, Webb AC, Dreyer
BE, et al. 1988 Identification
of a cDNA encoding
a pamthyroid
hormone-like
peptide
from a human hnnor associated
with humoral hypercalcemia
of malignancy.
Proc Nat1 Acad Sci USA. 85:597-601.
Suva LJ, Winslow
GA, Wettenhall
REH, et al. 1987 A parathyroid
hormonerelated
protein
implicated
in m&qxmt
hypercalcemi~x
cloning
and expression. Science.
237~893-896.
Broadus
AE, Stewart
AF. 1994 Parathyroid
hormone-related
protein:
structure, processing,
and physiological
actions. In: Bilezikian
Jl’, Marcus R, Levine
MA, eds. The pamthyroids:
basic and clinical
concepts.
New York: Raven
Press; 259-294.
Grill
V, Ho P, Body JJ, et al. 1991 Para0lyroid
hormone-related
protem:
elevated
levels in both humoral
hypercalcemi~
of malignancy
and hypercalcemia complicating
metastatic
breast cancer.
J Clin Endocrinol
Metab.
73:
1309-1315.
Fukumoto
S, Matsumoto
T, Watanabe
T, Takahashi
H, Miyoshi
1, Ogata E.
1989 Secretion
of parathyroid
hormone-like
activity
from human T-cell lymphotropic
virus type I-infected
lymphocytes.
Cancer Res. 4Y:384Y-3852.
Thiede
MA, Rodan
GA. 198X Expression
of a calcium-lnobili~ilg
parathyroid
hormone-like
peptide
in lactating
mammary
tissue. Science.
242:27X-280.
Moniz
C, Burton
PBJ, Malik
AN, et al. 19YO I’nrathyroid
hormone-related
peptide
in normal
human fetal development.
Mel Endocrinol.
5:25Y-266.
Kalaplis
AC, Luz A, Glowacki
J, et al. 1YY4 Lethal skeletal
dysplbsid
from
targeted
disruption
of the parathyrvid
liorlnone-reLtrd
peptidc
gene. Genes
Dev. 8:277-289.
Li X, Drucker
DJ. 1994 Parathyroid
hormone-r&&d
peptide
is d downstream
target for ms and SIC activation.
J Biol Chem. 269:6263-6266.
Watanabe
T, Yamaguchi
K, Takatsuki
K, Osame M, Yoshida
M. 1990 Constitutive
expression
of parathyroid
hormone-related
protein
gene in human T
cell leukemia
virus type I (HTLV-I)
carrws and adult T cell leukemia
patients
that can be transactivated
by HTLV-I
tax gene. J Exp Med. 172:75Y-765.
Holt EH, Vasavada
RC, Bander NH, Broadus
AE, Philbrick
WM. 1993 Regionspecific
methyl&ion
of the pxathyroid
hormone
related
peptide
gene determines its expression
in human renal carcinoma
cell hnes. J Biol Chem. 268:
20639-20645.
Jiippner
H, Abou-Samra
AB, Freeman
M, et al. 1991 A G protein-linked
receptor
for parathyroid
hormone
and px&hyroid
hormone-related
peptidc.
Science.
254~1024-1026.
Abou-Samra
AB, Jiippner
H, Force T, et al. 1YY2 Expression
cloning
of a
common
receptor
for pamthyroid
hormone
and parathyroid
hormone-related
peptide
from rat osteoblast-like
cells: a single receptor
stimul&es
intracellulx
accumulation
of both CAMP and inositol
trisphosphates
and increases
intracellular
free calcium.
Proc Nat1 Acad Sci USA. 8Y:2732-2736.
Schipani
E, Karga H, Karaplis
AC, et al. lYY3 Identical
complemmt~wy
deoxyribonucleic
acids encode
a human renal and bone parathyrold
hormone
(I’TH)/PTH-related
peptide
receptor.
Endocrnwlogy.
132:2157-2165.
Mangin
M, Ikeda K, Dreyer
BE, Broadus
AE. 1990 Identification
of an upstream promoter
of the humnn
pamthyroid
hormone-related
prptide
gene.
Mol Endocrinol.
4851-858.
Chilco
PJ, Gerardi
JM, Kaczmarczyk
SJ, Chu S, Leopold
V, Zajac JD. 1993
Calcitonin
increases
transcription
of parathyrvid
hormone-related
protein
via
cyclic AMP. Mol Cell Endocrinol.
94:1&7.
Insogna
KL, Stewart
AF, Morris
CA, Hough
LM, Nilstone
LM, Centrella
M.
1989 Native
and synthetic
analogue
of the malignancy-associated
parathyroid
hormone-like
protein
have in vitro transforming
growth factor-like
properties.
J Clin Invest. 83:1057-1060.
Centrella
M, Canalis
E, McCarthy
TL, Stewart AF, Orloff JJ, Insogna
KL. 1989
Parathyroid
hormone-related
protein
modulates
the effect of transforming
growth
factor-p
on deoxyribonucleic
acid and collagen
synthesis
in fetal rat
bone cells. Endocrinology.
125:1YY-208.
Burton
PBJ, Moniz
C, Knight
DE. 1990 I’arnthyroid
hormone
related peptide
can function
as an autocrine
growth
factor in human
renal cell carcinoma.
Biochem
Biophys
Res Commun.
167:1134-l
138.
Lange B, D’Angio
G, Ross 111 AJ, O’Neill
Jr JA, Packer RJ. lYY3 Oncologic
emergencies.
In: Pizo PA, Poplack
DG, eds. Principles
and practice
of pediatric oncology,
2nd ed. Philadelphia:
Lippincott;
951-972.
Kasahara
H, Tsuchiya
M, Addchi
R, Horikawa
S, Tanaka
S, Tachibana
S.
lYY2 Development
of a C-terminal-region-specific
radioinlnlunodssdy
of par+
thyroid
hormone-related
protein.
Biomed
Res. 13:155-161.
PTH-rP
PRODUCTION
H, Yamaoka
K, lshida
M, Yabuuchi
H. 1987 Circulating
22. Seino Y, Tanaka
lu,25-dihydroxyvitamin
D levels after a single dose of lcx,25-dihydroxyvitamin D, or lol-hydroxyvitamin
D3 in normal
men. Bone Miner.
2479-485.
23. Chomczynski
l’, Sacchi N. 1987 Singk+step
method
of RNA isolation
by acid
guanidium
thiocyanate-phenol-chroloform
extraction.
Anal
Biochem.
162:156-159.
24. Yamato H, Nagai Y, lnoue
D, et al. 1995 In vim evidence
for progressive
activation
of parathyroid
hormone-related
peptide
gene transcription
with
tumor growth and stimulation
of osteoblastic
bone formation
at an early stage
of humoral
hypercalcemia
of cancer. J Bone Miner Res. 10:36-44.
25. lkeda K, Ohno H, Hane M, et al. 1994 Development
of a sensitive
two-site
immunoradiometric
assay for parathyroid
hormone-related
peptide:
evidence
for elevated
levels in plasma
from patients
with adult T-cell leukemia/lymphoma and B-cell lymphoma.
J Clin Endocrinol
Metab. 79:1322-1327.
75th ANNIVERSARY:
on Diabetes
1095
26. Coffin
CM, Jaszcz W, O’Shea
PA, Dehner
LP. 1994 So-called
congenitalinfantile
fibrosarcoma:
does it exist and what is it? Pediatr
Pathol.
14~133-150.
27. Coffin
CM, Dehner
LP. 1990 Soft tissue tumors
in first year of life: a report
of 190 cases. Pediatr
Pathol.
10~509-526.
28. Wu TL, lnsogna
KL, Hough
LM, Milstone
L, Stewart
AF. 1987 Skin-derived
fibroblasts
respond
to human
parathyroid
hormone-like
adenylate
cyclasestimulating
proteins.
J Clin Endocrinol
Metab. 65:105-109.
29. Kremer
R, Karaplis
AC, Henderson
J, et al. 1991 Regulation
of parathyroid
hormone-like
peptide
in cultured
normal
human keratinocytes.
J Clin Invest.
87:884-893.
30. Inoue D, Matsumoto
T, Ogata E, Ikeda K. 1993 22-Oxacalcitriol,
a normocalcemic
analogue
of calcitriol,
supresses
both cell proliferation
and parathyroid hormone-related
peptide
gene expression
in human T cell lymphotrophic
virus, type l-infected
cells. J Biol Chem. 26&X16730-16736.
CELEBRATING
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Focusing on Beta-Cell Function
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l
Joslin Diabetes Center; Boston,
l
Karolinska Institute, Stockholm,
Call for Abstracts
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