平成 16 年度研究報告
腎細胞癌 (RCC) における副甲状腺ホルモン関連
タンパク (PTHrP) の産生
－ RCC と PTHrP －
研究テーマ
埼玉医科大学泌尿器科
助教授 上野 宗久
現所属：埼玉医科大学国際医療センター泌尿器腫瘍科
✻ サマリー ✻
副甲状腺ホルモン関連タンパク (parathyroid hormone-related protein, PTHrP) は副甲状腺ホルモ ンと N 末端が共通であり、副甲状腺ホルモン受容体と結合することにより、骨と腎臓において 副甲状腺ホルモン様の作用を発現する。腎細胞癌の全体の約 3％に Humoral hypercalcemia of malignancy と呼ばれる高カルシウム血症が腫瘍随伴症候群として認められ、これは PTHrP の過 剰産生に起因する。PTHrP 遺伝子は第 12 染色体短腕に位置し、9 つの exon で構成されており、 exon 5 と exon 6 はすべての PTHrP の転写に認められている。3’側の alternative splicing により、 3 つの異なる転写が行われ、これらの mRNA から各々C 末端領域が異なる 139 個、173 個と 141 個のアミノ酸からなるポリペプチドが生成されることが知られている。
今回、我々は 49 症例の腎細胞癌 (32 clear cell, 3 granular cell, 2 spindle cell, 1 collecting duct carcinoma, and 11 mixed) の摘出標本から得られた癌組織と正常腎組織から mRNA を分離し (Table 1)、all transcript と上記の 3 つの PTHrP の isoform について RT-PCR 法を施行した (Figure 1)。49 例の腎癌細胞の中で 48 例 (98％) に PTHrP が検出され、正常腎細胞においても 6 例 (6％) に検出された。最も高頻度の発現したのは 141 アミノ酸の isoform であり、139 アミノ酸、 173 アミノ酸の順に認めた。次にリアルタイム PCR 法で癌細胞と正常細胞における PTHrP の発 現を定量分析したところ (Figure 2)、癌細胞において有意により多くの PTHrP 産生を認め、そ の傾向は淡明細胞癌において顕著に見受けられた (Table 2)。高カルシウム血症を随伴したのは 1 例に過ぎず、PTHrP 産生と血中カルシウム濃度に相関は認められなかった。また、細胞異型 度 (grade) との間にも相関関係を見出せなかった。最後に、癌細胞において 139 アミノ酸の isoform 陽性例が陰性例よりも有意に多い PTHrP を産生することが示唆された (Table 2)。 腎摘標本のパラフィン包埋切片を PTHrP の C 末端に対する抗体で染色したところ、癌細胞すべ てに PTHrP 染色を認めたが、一部の正常腎細胞も非特異に染まり、免疫組織染色を用いたタン パクレベルでの解析は困難という結論が得られた。 以上より、正常腎細胞に比べて、淡明細胞を中心とした癌細胞がより多くの PTHrP mRNA を発 現することが判明した。更に腎癌細胞における PHTrP の産生には 139 アミノ酸の isoform 陽性 率が深く関与していることが示唆された。
✻ 研究報告 ✻
OBJECTIVES
Parathyroid hormone-related protein (PTHrP) shares some amino-terminal sequence identity with parathyroid hormone (PTH) and binds to a common receptor (PTH/PTHrP receptor), producing classical PTH-like effects in bone and the kidney. PTHrP was originally isolated, cloned, and sequencedfrom tumors associated with hypercalcemia and has been identified in a variety of adult and fetal tissues. The human PTHrP gene is located on the short arm of chromosome 12 and consists of nine exons, of which two are present in all PTHrP transcripts: exon V, encoding the prepro region of the protein, and exon VI, which encodes the mature protein up to residue 139 (Fig. 1A). Via alternative 3’ splicing, three different transcripts can be formed. The polypeptides encoded by these three mRNAs have been deduced to consist of 139, 173, and 141 amino acid isoforms, respectively, which differ in their C-terminal regions. However, it has not been determined how these differing C-terminal regions may act biologically and thus which isoforms may play a key role in the development of hypercalcemia in patients. Renal cell carcinoma (RCC) accounts for approximately 3% of all patients with humoral hypercalcemia of malignancy, which is believed to be caused by excessive PTHrP production.
In the present study, we compared the mRNA expression of PTHrP in malignant tissues to that in non-malignant tissues of the patients with RCC. We also detected the roles of the histological types of RCC and the 3’ splice variants to produce PTHrP.
METHODS
Patients
All the specimens were obtained from nephrectomy of 49 patients with renal cell carcinoma (32 clear cell, 3 granular cell, 2 spindle cell, 1 collecting duct carcinoma, and 11 mixed, Table 1). There were 16 females and 33 males, aged 21-80 years (mean 62.1 years). Tumor tissue samples were obtained in accordance with Helsinki Declaration.
Purification of total RNA
The tumor specimen was cut into pieces. Total RNA was extracted from tumor samples according to the protocol of the RNeasy total RNA reangent set (Qiagen). The RNA content was estimated by spectroscopy on a GeneQuant II spectrophotometer (Pharmacia) and by gel analysis. The purity was estimated by the ratio of the absorbance at 260/280. The RNA was stored at -80˚C until use.
RT-PCR
The RNA was reverse-transcribed using the GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR Kit (PE Biosystems). For first-strand cDNA synthesis, 500 ng of total RNA from each sample and PTHrP reverse primer (Fig. 1B) were incubated with rTth polymerase at 70˚C for 10 min. For PCR, the product was incubated with PTHrP forward primer (Fig. 1B) at 40 cycles of 95˚C for 30 s, 55˚C for 30 s,
and 72˚C for 1 min, followed by a final cycle at 72˚C for 10 min. After PCR, aliquots of the reaction were analyzed on a 1.2% agarose gel with ethidium bromide (0.5 mg/ml).
Real-time PCR
The primers used in the analysis of PTHrP gene expression are given in Fig. 2A. The PCR reactions were performed in a LightCycler apparatus using SYBR Green 1. To correct the differences in both RNA quality and quantity between samples, data were normalized using the ratio of the target cDNA concentration to that of GAPDH. The published GAPDH primer pair was used. The fluorescence of individual samples was measured by the Lightcycler at the end of every cycle. The Lightcycler software algorithm automatically determined the crossing point (Cp) for the individual samples including the calibrators (Fig. 2B). Calibration curves were constructed by plotting the Cp vs the number of copies for each calibrator (Fig. 2C). The numbers of copies in unknown samples were established by comparing their Cps with the calibration curve.
Statistical analysis
Differences between the means of the analyzed variables observed in the kidney samples were determined by the Mann-Whitney nonparametric U-test. P＜ .05 (two-tailed) was considered significant.
RESULTS
#1 PTHrP transcripts were found in 48 out of the 49 RCC specimens (Table 1).
#2 The expression of PTHrP mRNA significantly increased in the carcinoma specimens compared to that in normal kidney tissues (Table 1, 2).
#3 The malignant tissues containing clear cells seem to mainly produce PTHrP (Table 2). #4 The 141-amino acid PTHrP was the most abundant of the 3’ splice variants in RCC (Table 2).
#5 The mRNA encoding the 139-amino acid variant had higher PTHrP levels than that not encoding 139-amino acid variant (Table 2).
CONCLUSIONS
RCC, and in particular clear cell carcinoma, produces PTHrP to a greater extent than does the normal kidney. Positivity for PTHrP 139-amino acid mRNA expression seems to be correlated with the amount of PTHrP production.
