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Experimental Studies |
Department of Urology, Asahikawa Medical College, Asahikawa, Hokkaido 078, Japan
Address all correspondence and requests for reprints to: Dr. Satoshi Yamaguchi, Department of Urology, Asahikawa Medical College, Nishikagura 4–5-3–11, Asahikawa, Hokkaido 078, Japan.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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The specimens used for the study were 42 parathyroid glands from 21 patients with primary hyperparathyroidism (19 cases of adenoma and 2 cases of primary hyperplasia due to multiple endocrine neoplasia type 1) and 129 parathyroid glands from 32 patients with secondary hyperparathyroidism. An additional 40 parathyroid glands resected during thyroid surgery of 30 normocalcemic patients were used as normal controls.
In normally functioning parathyroids, a small number of cells in the growth phase were found. In primary hyperparathyroidism, proliferative activity was highest in the adenoma followed by primary hyperplasia. In contrast, the PCNA LIs showed a low value in the normal rim of the adenoma and normal glands resected as biopsy specimens from adenoma patients. We, therefore, assumed that proliferative activity was suppressed in these cells compared with that in normally functioning glands. In secondary hyperparathyroidism, when the cell component of the parathyroid tissues was divided into five types, PCNA immunoreactivity was lowest in the dark chief cells. Proliferative activity in cells of the oxyphil series was the same or higher than that in the clear chief cells or vacuolated chief cells. When classified according to the structure of the parathyroid glands, cell proliferation was significantly higher in the nodular type than in the diffuse type (maximum LI, 176 ± 231 vs. 38.3 ± 55.7; average LI, 120 ± 188 vs. 24.8 ± 43.5; mean ± SD; P < 0.001). More PCNA-immunoreactive cells were found in autotransplanted glands with recurrence than in glands resected during the initial surgery. To summarize the PCNA expression classified according to the pathological types of hyperparathyroidism, the PCNA LIs were highest in secondary hyperplasia (maximum LI, 144 ± 212; average LI, 96.0 ± 169) and adenoma (maximum LI, 102 ± 81.7; average LI, 67.5 ± 67.7), followed by primary hyperplasia (maximum LI, 25.0 ± 25.4; average LI, 19.2 ± 22.2) and normal glands (maximum LI, 13.6 ± 23.9; average LI, 4.40 ± 8.90).
These findings suggest that the cellular proliferative kinetics of the parathyroid gland differ depending on the type of hyperparathyroidism, glandular structure, and cell components. As the detection method of intranuclear expression of PCNA in cells is too sensitive, we should be careful not to overestimate the number of cells in the proliferative cycle. However, these results could not have been obtained using a conventional method such as DNA analysis by flow cytometry.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The conventional way to examine cellular kinetics in hyperparathyroidism has been to analyze DNA quantity by flow cytometry. Discussions were based mainly on the degree of aneuploidy in the DNA histogram (1). However, in examinations by the ploidy pattern of DNA quantity, the reported frequency of aneuploidy in normal parathyroid, adenoma, hyperplasia, and carcinoma differs between studies, presumably due to the lack of a uniform method and definition for flow cytometric analysis, particularly for dispersion and stain techniques (1). Therefore, in the present study, we attempted to estimate cellular proliferative activity by an immunohistochemical method, which does not use flow cytometric equipment.
Proliferating cell nuclear antigen (PCNA) is a 36-kDa acidic nuclear
protein that has been very highly conserved in the process of evolution
and is a cell proliferation marker that is recently drawing attention.
PCNA functions as a cofactor for DNA polymerase 
, which is required
for both DNA replication and DNA repair (2, 3). PCNA is mainly
synthesized during the G1 phase of the cell cycle, and its amount is
maximum during the S phase (4). Therefore, by observing PCNA expression
in the cell nuclei, it is possible to know the proliferative activity
of the cells. As PCNA does not require any particular pretreatment and
can be applied to a wide range of species, and as analysis using
ordinary pathological specimens, such as methanol- or formalin-fixed,
paraffin-embedded sections is possible (5, 6), this method is thought
to be suitable for retrospective examination of samples preserved in
the past. As the anti-PCNA monoclonal antibody was created in 1987,
there have been many studies using PCNA for judging biological
malignancy and examining the clinical outcome of various types of
neoplasms (6). However, there are few reports (7, 8) that have examined
PCNA in connection with benign diseases. We examined PCNA expression in
parathyroid cells in hyperparathyroidism and estimated the
proliferative kinetics of parathyroid cells for various conditions of
hyperparathyroidism.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Primary hyperparathyroidism. The specimens were 42 parathyroid glands obtained from 21 patients who underwent parathyroidectomies between 1984 and 1993. These included 19 adenomas, 16 normal parathyroid glands removed for biopsy specimens during adenoma resection, and 7 primary hyperplasias [2 cases of multiple endocrine neoplasia (MEN) type 1]. The clinical findings that led to the discovery of hyperparathyroidism were 10 cases of urolithiasis type (including 1 case of MEN type 1) and 11 cases of chemical type (including 1 case of MEN type 1). No bone-type hyperparathyroidism or parathyroid carcinoma was experienced during the period of this study.
Secondary hyperparathyroidism. Between 1987 and 1994, 37 parathyroid operations were performed on patients with secondary hyperparathyroidism who displayed symptoms of severe renal osteodystrophy that resisted medical treatment. The patients had been undergoing long term hemodialysis treatment due to chronic renal failure. Of the 136 parathyroid glands resected in surgery, 129 were used in our study, excluding the seven glands that were used up completely for autotransplantation and, therefore, were not available for pathological examination. The parathyroid glands included 118 glands from 32 patients who had undergone total parathyroidectomy and autotransplantation to the arm in initial surgery, 10 glands from 4 patients who had undergone graftectomy for recurrent hyperparathyroidism, and 1 gland from a patient who had undergone parathyroidectomy for persistent hyperparathyroidism due to intramediastinal supernumerary gland.
Normal control.
The specimens for the normal control were 40
parathyroid glands resected during total thyroidectomy with lymph node
dissection due to advanced thyroid cancer in 30 patients performed
between 1980 and 1991. The existence of hyperparathyroidism in these
patients was denied because serum calcium and inorganic phosphorous
levels were within the normal range in the preoperative examination.
Patient characteristics are shown in Table 1
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Evaluation of staining
The PCNA-positive reaction was noted within the cell nuclei, and
the cells whose nuclei were clearly brown from the
3,3'-diaminobenzidine hydrochloride instead of green from the methyl
green were interpreted as positive. The following steps were taken to
evaluate the staining results. We selected two areas: one where the
largest number of PCNA-positive cells was present, and another where
PCNA-positive cells were randomly distributed other than the maximal
stained area. Microphotographs of these areas were taken at a
magnification of x150 or x300. From these photographs, we defined the
PCNA labeling index (LI) as the number of PCNA-positive cells per 1000
parathyroid cells and used it for various analyses. As it was expected
that the PCNA LI would differ depending on the cells comprising the
parathyroid, the PCNA LI was calculated for each of the cell components
with a measurement area of adequate size. More specifically, we counted
1000 parathyroid cells in at least 2 areas of each region of interest,
and the average of the number of PCNA-positive cells in the area where
they existed in the largest number was obtained as the maximum LI (Fig. 1A), and their number in the area where they were
distributed evenly was obtained as the average LI (Fig. 1B), similar to
those described by Carroll et al. (9). By using hematoxylin
and eosin stain on the specimens corresponding to the above
immunohistochemical stains, the cell components were classified into
the following five types: dark chief cell with densely distributed
nucleus, small cytoplasm, and a dark oval appearance; clear chief cell,
which is larger in size and has a cytoplasm with a brighter appearance;
vacuolated chief cell, which is even larger and has a slightly smaller
nucleus and a large vacuole within the cell; oxyphil cell, which is the
largest in size, with a large nucleus and a cytoplasm that shows an
eosinophilic stain; and transitional oxyphil cell, which is somewhat
smaller than the oxyphil cell and whose cytoplasm shows a lighter shade
when stained with eosin. As a result of this classification, we needed
to examine 60 regions for primary hyperparathyroidism, 283 regions for
secondary hyperparathyroidism, and 43 regions for the normal control.
Furthermore, the glandular structure, composed of these 5 types of cell
components in secondary hyperparathyroidism, was classified into 2
types: the diffuse type, which shows a diffuse growth pattern while
maintaining a normal tissue structure, and the nodular type, which
consists of various sizes of nodules well demarcated from the
surrounding fibrous tissue.
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Statistical significance was calculated using a nonparametric test (Wilcoxon-Mann-Whitney test) in the case of a single comparison. For multiple comparisons, Scheffe’s post-hoc test was used only if one-way factorial ANOVA showed a significant difference. The correlation coefficient and its significance were calculated using Spearman’s rank correlation. Statistical signification was assumed at P < 0.05. All results were expressed as the mean ± SD.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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For the normal control, we examined 40 glands at 43 regions. The
detection rate of PCNA-positive cells was 27 of 40 glands (68%), and
the normally functioning parathyroid was found to contain cells in the
proliferative phase. The PCNA LIs were: maximum LI, 13.6 ±
23.9/103 cells; and average LI, 4.40 ±
8.90/103 cells. PCNA LIs for the three types of cell
components were determined (Table 2). In summary, the
relationship among the PCNA LIs was clear chief cell > dark chief
cell > oxyphil cell, but no significant difference was noted
among the three. Also, there was no correlation between age and the
PCNA LIs.
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For primary hyperparathyroidism, we analyzed the PCNA LIs of 19
adenomas (20 regions), 7 hyperplasias (7 regions), 17 regions of the
normal rim of adenomas, and 16 glands (16 regions) of normal
parathyroid glands obtained as biopsy specimens during adenoma
resection (Table 2
). The breakdown by component cells were: for
adenoma, 19 regions of clear chief cells, 1 region of oxyphil cells;
for hyperplasia, 7 regions of clear chief cells; for the normal rim, 7
regions of dark chief cells, 10 regions of clear chief cells; and for
the normal parathyroid gland obtained as a biopsy specimen, 10 regions
of dark chief cells, 6 regions of clear chief cell. The detection rates
of PCNA-positive cells were 19 of 19 (100%) for adenoma (Fig. 2A), 7 of 7 (100%) for hyperplasia, 10 of 17 (59%) for
the normal rim of adenoma (Fig. 2B), and 9 of 16 (56%) for the
parathyroid gland obtained as a biopsy specimen. In comparing the PCNA
LIs, both the maximum LI and the average LI were the highest for
adenoma, followed by hyperplasia, normal rim of adenoma, and then
parathyroid gland obtained as a biopsy specimen (Table 2). Also,
compared with the normally functioning parathyroid gland, PCNA LIs were
significantly higher for adenoma (P < 0.01, maximum LI
and average LI) and hyperplasia (P < 0.05, average
LI), and lower for the normal rim of adenoma and the parathyroid gland
obtained as a biopsy specimen (P < 0.05, maximum LI;
Table 2).
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For secondary hyperparathyroidism, the PCNA LIs for 129 glands
(283 regions) were examined. The glands included 118 glands (266
regions) obtained during parathyroidectomy performed as the initial
operation, 10 glands (14 regions) resected due to autotransplanted
graft recurrence, and 1 gland (3 regions) resected due to recurrence of
intramediastinal ectopic supernumerary gland. The detection rate of
PCNA-positive cells was 128 of 129 glands (99%); the only gland in
which no positive cells were detected was that obtained at initial
surgery showing a diffuse growth pattern. In the examination by
component cells, 21 regions of dark chief cells, 94 regions of clear
chief cells, 39 regions of vacuolated chief cells, 80 regions of
transitional oxyphil cells, and 49 regions of oxyphil cells were
compared. In comparing each PCNA LI, the dark chief cell’s LIs was
significantly lower than that of other component cells (Table 2).
In the examination by glandular structure, 27 diffuse-type glands (49
regions) and 91 nodular-type glands (217 regions), excluding recurrent
cases, were examined. The PCNA LIs were significantly higher for the
nodular type (Fig. 3). Classifying by the type of
glandular structure of the parathyroid resected from a single
patient, 2 cases (8 glands) showed diffuse growth only, 16 cases (60
glands) showed nodular growth only, and 14 cases (50 glands) showed a
combination of both. The PCNA LIs were examined for the combined cases,
namely 19 diffuse-type glands (36 regions) and 31 nodular-type glands
(84 regions), to determine the difference in the proliferative activity
between the 2 in the same individuals. The comparison in the
parathyroid obtained from a single patient also indicated that both
PCNA LIs were significantly higher for the nodular type than the
diffuse type (maximum LI, 214 ± 279 vs. 35.6 ±
47.9; average LI, 160 ± 245 vs. 22.4 ± 35.9;
P < 0.001).
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). In 2 cases, it was actually possible to compare the
tissues originating in the same parathyroid obtained from initial
surgery and graftectomy (In the other 2 cases, the parathyroid had
been used up completely for autotransplantation at parathyroidectomy
and, therefore, could not be examined). In both cases, the PCNA LIs had
increased after recurrence compared with the time of initial surgery,
with the maximum LI and the average LI rising from 87.0 to
228/103 cells (2.6 times increase) and from 51.0 to
115/103 cells (2.3 times increase), respectively, in the
first case and with these values rising from 34.0 to
418/103 cells (12.3 times increase) and from 22.0 to
348/103 cells (15.8 times increase), respectively, in the
second case. Also, compared with the PCNA LIs of the parathyroid glands
resected during the initial surgery, no significant difference was
noted between the recurrent and nonrecurrent cases.
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In comparing the PCNA LIs for the different pathological types,
the order from highest to lowest was secondary hyperplasia, adenoma,
primary hyperplasia, and normally functioning gland (Table 2).
Statistically significant differences were noted, except between
secondary hyperplasia and adenoma. There was a good correlation between
maximum LI and average LI in this series (n = 386; r2
= 0.939; P < 0.001).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Immunohistochemical procedures for evaluating cell proliferation have been reported recently, and the merits of PCNA analysis have been shown (5, 6). PCNA is an intranuclear protein that mainly appears between the G1 phase and the M phase (proliferative phase) of the cell cycle, and especially between the late G1 phase and the S phase. Bromodeoxyuridine (BrdU) and Ki-67 are also representative cellular proliferation markers, like PCNA, but both have some disadvantages. BrdU is a thymidine analog and needs to be incubated into the fresh specimen beforehand. In vitro, proliferative cells are identified by immunohistochemical staining using anti-BrdU antibody after the BrdU has infiltrated cells in the S phase. However, ethical problems arise in an in vivo investigation because BrdU must be administered directly into the body (6). Ki-67 is a nuclear nonhistone protein that appears in the proliferating cycle other than the G0 phase. Recently, MIB-1 antibody has been available in formalin-fixed paraffin-embedded section (14). However, microwave treatment is necessary in MIB-1 staining, and the subject tissues sometimes detach from the glass slide during this process (15). The biggest advantage of using PCNA for analysis is that no complex pretreatment is required; therefore, proliferating cells can be detected with relative ease from histopathological specimens preserved by ordinary methods.
In primary hyperparathyroidism, the PCNA LIs were highest in adenomas, followed by primary hyperplasias. In both cases, the PCNA LIs were significantly higher than those in normally functioning parathyroid, and it seemed that cellular proliferative activity was accelerated in the order of primary hyperplasia and then adenoma. Meanwhile, PCNA LIs in the normal rim of adenomas and in parathyroid glands other than adenomas were significantly lower than those in adenomas and primary hyperplasias, and even lower than those in normally functioning glands. Loda et al. (15) reported that they found no PCNA-positive cells in the normal rim of adenomas or in parathyroids other than adenomas, but in our study, positive cells, although few in number, were present in both cases. This led us to the assumption that cellular proliferative activity is not suspended in the normal rim of adenomas and parathyroid glands other than adenomas, but, rather, is in a low or suppressed condition.
In secondary hyperparathyroidism, it has been reported that there is no difference in cellular proliferative activity depending on the cell component type (15, 16). However, our results showed that cellular proliferative activity is lowest in dark chief cells. In an ultrastructural study, Shannon and Roth (17) reported that chief cells have a secretory cycle, in which they change from the resting phase (large cells) to the packaging phase (small cells). Perfitt (18) showed a schema in the relationship between the proliferative cycle and the secretory cycle, indicating that the dark chief cells are most active in hormone secretion, but are in the G0 phase in the proliferative cycle. It is possible that the clear chief cells change from the G0 to the G1 phase in the proliferative cycle. This concept is in agreement with our results, which indicated that cell proliferation was lowest in the dark chief cells. It is known that oxyphil cells, along with vacuolated chief cells and transitional oxyphil cells, occupy a larger portion of the parathyroid tissue in secondary hyperparathyroidism (19). However, there has been no report on the proliferative activity of each of these cells. Our results suggest that cells of the oxyphil series have the same or higher proliferative activity as clear chief cells or vacuolated chief cells. In addition, the proliferative potential of the transitional oxyphil cell, which is considered to be an intermediate type of chief cells and oxyphil cells (19), was higher than that of the oxyphil cell, indicating that cellular proliferative kinetics may be transformed according to changes in cellular morphology.
Our analysis of the glandular structure indicated that cellular proliferative activity was higher in the nodular type than in the diffuse type, which agrees with past reports (15, 16). Ohta et al. (16) examined the distribution of PCNA-positive cells in the individual nodules of the nodular-type gland and reported that more proliferating cells were found in the peripheral region than in the central region of the nodule. In the present study, we also noted several locations in the peripheral region where a large number of positive cells were present. When tumorous proliferation occurs in an organ, it is quite natural for active cells to multiply in the peripheral regions, and it may be that the relative decline in proliferative activity in the central region is due to a lack of feeding vessels.
As recurrence in grafted glands occurs more frequently in the nodular type than in the diffuse type, Tominaga et al. (20) have reported that the cellular proliferative activity of an autograft may be influenced by the original proliferative ability of the glands before grafting. However, in our study, even in the same nodular-type glands, cellular proliferative activity was significantly higher in the autograft with recurrence than in the gland resected at initial surgery. This indicates that there is a possibility that the continuous stimulation due to chronic renal failure may cause the cellular proliferative kinetics of the parathyroid to change.
The number of cells in the proliferative cycle estimated by PCNA LI is fairly larger than that shown in the published works (21, 22) on parathyroid cell proliferation. This difference can be explained as follows. 1) As PCNA emerges in the G1–M phases, the range recognized with this PCNA method in the cell cycle is broader than that with [3H]thymidine that is incorporated in S phase or the mitotic index that indicates M phase. 2) In the analysis of cell cycle marker using an immunohistochemical method, PCNA can be detected even in a trace amount, and the present PCNA method is much more sensitive than the conventional method. Of course, we should be careful to avoid possible overestimation of the presence of PCNA, because the long half-life of PCNA (23) leads to labeling of cells that have already left the cell cycle (24), PCNA may be induced in normal noncycling cells adjacent to tumors (5), and PCNA exists at low level in noncycling cells during DNA repair synthesis (4, 25). Comparisons to other proliferative markers, such as Ki-67 (15) and silver staining nucleolar organizer regions (4), are needed to confirm the accuracy of this method.
It is known that parathyroid cells and tumors grow very slowly (18). In this respect, the high proliferative potential of parathyroid cells and the growth rate of tumors shown in this study appear to be contradictory. This discrepancy can be explained if cells proliferate on one side and die due to apoptosis on the other. However, this was not confirmed by the rat experiment (26). Uda et al. (27) reported that the number of Ki-67-positive cells was large, but that of apoptotic cells was small; on the other hand, expression of Bcl-2 protein, which is an inhibitor of apoptosis, was increased in human parathyroid glands with secondary hyperparathyroidism. These results indicate that the relation among the growth of parathyroid tumor, cell-proliferative potential, and apoptosis is not simple. Further examination needs to be performed.
Recently, parathyroid tumors are actively examined molecular biologically, and there is a report (3) indicating the relation to PCNA. Candidate genes of MEN type 1 are known to exist in chromosome 11q13 (28, 29), and it has been revealed that PRAD1, a putative oncogene, exists in this region. On the other hand, in a chromosomal inversion event deduced in a subset of parathyroid adenomas, tumor-specific DNA rearrangements juxtapose the 5'-regulatory region of PTH gene, which is normally located on chromosome 11p15, with the coding region of PRAD1 (30). This leads to dramatic overexpression of the PRAD1 protein, commonly called cyclin D1, controlling the cell cycle (31). Cyclin D1, occurring at a critical point in the mid- to late G1 phase, forms a complex with PCNA inside the nuclei and is believed to have close connections with PCNA (3). Meanwhile, with the MEN type 1 gene, it has been suggested that a monoclonal overgrowth of cells occurs due to the loss of heterozygosity of the tumor suppressor gene, which is estimated to exist in the same chromosome 11 region from PYGM to INT-2 (32). In the study of monoclonarity of secondary and primary parathyroid hyperplasia, Arnold et al. (33) found loss of heterozygosity at the M27ß region of the X-chromosome. They came to the conclusion that inactivation of a novel X-chromosome tumor suppressor gene may lead to monoclonal overgrowth of parathyroid tumors. These reports indicate that other mechanisms may play a role in controlling parathyroid cell proliferation.
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Acknowledgments |
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Footnotes |
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Received January 18, 1996.
Revised June 17, 1996.
Revised December 19, 1996.
Accepted February 27, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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