Malignant transformation of the human endometrium is associated with overexpression of lactoferrin messenger RNA and protein.
D K WalmerC. PadinM A WronaB E HealyRex C. BentleyMing‐Sound TsaoMatthew F. KohlerJohn A. McLachlanKaren Gray
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In the mouse uterus, lactoferrin is a major estrogen-inducible uterine secretory protein, and its expression correlates directly with the period of peak epithelial cell proliferation. In this study, we examine the expression of lactoferrin mRNA and protein in human endometrium, endometrial hyperplasias, and adenocarcinomas using immunohistochemistry, Western immunoblotting, and Northern and in situ RNA hybridization techniques. Our results reveal that lactoferrin is expressed in normal cycling endometrium by a restricted number of glandular epithelial cells located deep in the zona basalis. Two thirds (8 of 12) of the endometrial adenocarcinomas examined overexpress lactoferrin. This tumor-associated increase in lactoferrin expression includes an elevation in the mRNA and protein of individual cells and an increase in the number of cells expressing the protein. In comparison, only 1 of the 10 endometrial hyperplasia specimens examined demonstrates an increase in lactoferrin. We also observe distinct cytoplasmic and nuclear immunostaining patterns under different fixation conditions in both normal and malignant epithelial cells, similar to those previously reported in the mouse reproductive tract. Serial sections of malignant specimens show a good correlation between the localization of lactoferrin mRNA and protein in individual epithelial cells by in situ RNA hybridization and immunohistochemistry. Although the degree of lactoferrin expression in the adenocarcinomas did not correlate with the tumor stage, grade, or depth of invasion in these 12 patients, there was a striking inverse correlation between the presence of progesterone receptors and lactoferrin in all 8 lactoferrin-positive adenocarcinomas. In summary, lactoferrin is expressed in a region of normal endometrium known as the zona basalis which is not shed with menstruation and is frequently overexpressed by progesterone receptor-negative cells in endometrial adenocarcinomas.Keywords:
Lactoferrin
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SATO, S., JIKO, K., ITO, K., OZAWA, N., YAJIMA, A., MIYAZAKI, S. and SASANO, H. Expression of c-myc mRNA and Protein in Human Endometrial Carcinoma; Simultaneous Study of In Situ Hybridization and Immunohistochemistry. Tohoku J. Exp. Med., 170 (4), 229-234 - We examined the c-myc gene at both mRNA and protein levels in tumors of 10 endometrial carcinomas by simultaneous in situ hybridization and immunohistochemistry. Expression of c-myc mRNA was observed in all cases by in situ hybridization. Immunohistochemically, c-myc protein expression was also observed in all cases and immunoreactivity was recognized as nuclear staining. The site of c-myc protein expression was mostly consistent with that of c-myc mRNA expression. While there has been no clear correlation between the expression of c-myc mRNA and/or protein and prognostic parameters so far, this approach will contribute to an understanding of oncogene expression and its biological significance in human gynecological malignancies.
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To understand the function of SRG4, a novel spermatogenesis gene, we studied its expression pattern during normal mouse development and in experimental cryptorchidism. Testis tissues were collected from 1-, 3-, and 12-week-old normal mice and immunohistochemistry was used to detect the expression of SRG4. We performed surgery on mice to cre- ate unilateral cryptorchidism and monitored SRG4 mRNA levels by semi-quantitative RT-PCR in the cryptorchid testis from day 0-18. At post-operative day 18, the cryptorchid testis and the contralateral control testis were harvested and assayed for SRG4 expression by in situ hybridization. Immunohistochemistry results showed that SRG4 protein was hardly detected in 1-week-old mouse testis, but the expression was present in 3-week-old mouse and abundant in 12-week-old mouse testis. SRG4 immunostaining was mainly localized to the cytoplasm and membrane of spermatocytes and round spermatids. Moreover, semi-quantitative RT-PCR result showed the expression of SRG4 mRNA did not decrease until 9 d after cryptorchid surgery, and continued to decline thereafter. In situ hybridization revealed that in contrast to the abundant SRG4 expression in the control side, few remaining germ cells in the crytorchid testis were positive for SRG4 at d 18 after surgery. The results indicated that the expression of SRG4 was regulated by development, and SRG4 was mainly expressed in the cytoplasm of spermatocytes and round spermatids. However, in cryptorchid testis, in which most germ cells undergo apoptosis, only a few of SRG4 is observed, suggesting that SRG4 may be as a specific marker to evaluate the process of spermatogenesis.
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We examined the immunohistochemical localization of p53, a tumor suppressor gene, in thirty-three specimens of uterine endometrial carcinoma which included one case of carcinosarcoma, eleven specimens of endometrial hyperplasia and ten specimens of normal endometrium. We also analyzed the association between the immunolocalization of p53 protein and the clinical and pathological parameters of endometrial concern. We also determined whether p53 mRNA is overexpressed in these specimens by in situ hybridization and simultaneous immunohistochemistry and in situ hybridization. Immunohistochemistry was performed using the monoclonal antibodies pAbDO-7 and pAb1801, and polyclonal antibody pAbCM-1. Immunoreactive p53 was observed in the nuclei of the cancer cells in 15/33 (45%) by pAbDO-7, 11/26 (42%) by pAb1801, and 16/33 (48%) by pAbCM-1. No p53 immunoactivity could be detected in either hyperplasia or normal endometrium except for a case in which the endometrium was in the secretory phase. There was no significant relationship between p53 immunostaining as determined by pAbDO-7, and age, clinical stage, histological grade or depth of myometrial invasion. We employed the 35S-labeled antisense single stranded synthetic oligonucleotide probe, ON102, to perform in situ hybridization and simultaneous immunohistochemistry and in situ hybridization. In every case of endometrial carcinoma studied, no significant accumulation of the p53 hybridization signal was observed in carcinoma cells. This indicated that overexpression of p53, as observed by immunohistochemical staining, is not due to an increase in the steady-state level of p53 mRNA in carcinoma cells. These results suggest that immunostaining of p53 is associated with the malignant phenotype, but does not correlate with the biological behavior of human endometrial carcinoma.
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In the mouse uterus, lactoferrin is a major estrogen-inducible uterine secretory protein, and its expression correlates directly with the period of peak epithelial cell proliferation. In this study, we examine the expression of lactoferrin mRNA and protein in human endometrium, endometrial hyperplasias, and adenocarcinomas using immunohistochemistry, Western immunoblotting, and Northern and in situ RNA hybridization techniques. Our results reveal that lactoferrin is expressed in normal cycling endometrium by a restricted number of glandular epithelial cells located deep in the zona basalis. Two thirds (8 of 12) of the endometrial adenocarcinomas examined overexpress lactoferrin. This tumor-associated increase in lactoferrin expression includes an elevation in the mRNA and protein of individual cells and an increase in the number of cells expressing the protein. In comparison, only 1 of the 10 endometrial hyperplasia specimens examined demonstrates an increase in lactoferrin. We also observe distinct cytoplasmic and nuclear immunostaining patterns under different fixation conditions in both normal and malignant epithelial cells, similar to those previously reported in the mouse reproductive tract. Serial sections of malignant specimens show a good correlation between the localization of lactoferrin mRNA and protein in individual epithelial cells by in situ RNA hybridization and immunohistochemistry. Although the degree of lactoferrin expression in the adenocarcinomas did not correlate with the tumor stage, grade, or depth of invasion in these 12 patients, there was a striking inverse correlation between the presence of progesterone receptors and lactoferrin in all 8 lactoferrin-positive adenocarcinomas. In summary, lactoferrin is expressed in a region of normal endometrium known as the zona basalis which is not shed with menstruation and is frequently overexpressed by progesterone receptor-negative cells in endometrial adenocarcinomas.
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Occasional reports indicated cytokeratin (CK) protein expression (mainly by immunohistochemistry) in malignant melanoma (MM) and suggested an association with unfavorable clinical parameters. However, the mRNA expression of CK and its clinicopathologic significance in MM has not been specifically evaluated. We investigated the mRNA and protein expression of nine CKs in melanoma cell lines and tissues, in particular the prognostic significance of CK18 mRNA expression. Reverse transcription (RT)-PCR (CK6–10, 14 and 18–20), in-situ hybridization (ISH) (CK18), and western blotting (CK18 and pan-cytokeratin AE1/AE3) were performed on MM cell lines A375, A875, M14, and SK-MEL-1. Eighty MM tissue samples were analyzed by ISH and immunohistochemistry for CK18 expression. The mRNA of CK6–8, 10, 14, 18, and 19 (but not CK9 and 20) was detected in one to four of the melanoma cell lines by RT-PCR. CK18 was detected in all four cell lines by RT-PCR, ISH, and western blotting. CK18 mRNA ISH was positive in three of 30 (10.0%), 10 of 25 (40.0%), and 12 of 25 (48.0%) of primary cutaneous, primary mucosal, and metastatic melanomas, respectively (overall positivity: 25 of 80, 31.3%). CK18 immunostaining was only observed focally in eight of 80 (10.0%) of MM tissue samples, and AE1/AE3 immunostaining was altogether negative. Significantly, CK18 mRNA ISH positivity (but not protein immunohistochemistry) was associated with poorer prognosis by both univariate analysis (P<0.001) and multivariate analysis (relative risk=5.430, 95% confidence interval 2.246–13.128, P<0.001). CK18 mRNA could be identified in one-third of melanoma tissue samples and is an adverse prognostic factor. ISH is superior to immunohistochemistry for analyzing CK18 expression in MM.
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