For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, b-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, b-2-microglobin, b-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tumour progression and metastasis. In this study, we investigated the in vitro and in vivo expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA and protein in a previously described human melanoma xenograft model. This model consists of eight human melanoma cell lines with different metastatic behaviour after subcutaneous (s.c.) injection into nude mice. MMP-1 mRNA was detectable in all cell lines by reverse transcription polymerase chain reaction (RT-PCR), but the expression was too low to be detected by Northern blot analysis. No MMP-1 protein could be found using Western blotting. MMP-2 mRNA and protein were present in all cell lines, with the highest expression of both latent and active MMP-2 in the highest metastatic cell lines MV3 and BLM. MMP-3 mRNA was expressed in MV3 and BLM, and in the non-metastatic cell line 530, whereas MMP-3 protein was detectable only in MV3 and BLM. None of the melanoma cell lines expressed MMP-9. TIMP-1 and TIMP-2 mRNA and protein, finally, were present in all cell lines. A correlation between TIMP expression level and metastatic capacity of cell lines, however, was lacking. MMP and TIMP mRNA and protein expression levels were also studied in s.c. xenograft lesions derived from a selection of these cell lines. RT-PCR analysis revealed that MMP-1 mRNA was present in MV3 and BLM xenografts, and to a lesser extent in 530. Positive staining for MMP-1 protein was found in xenograft lesions derived from both low and high metastatic cell lines, indicating an in vivo up-regulation of MMP-1. MMP-2 mRNA was detectable only in xenografts derived from the highly metastatic cell lines 1F6m, MV3 and BLM. In agreement with the in vitro results, the highest levels of both latent and activated MMP-2 protein were observed in MV3 and BLM xenografts. With the exception of MMP-9 mRNA expression in 530 xenografts, MMP-3, MMP-9, and TIMP-1 mRNA and protein were not detectable in any xenograft, indicating a down-regulated expression of MMP-3 and TIMP-1 in vivo. TIMP-2 mRNA and protein were present in all xenografts; interestingly, the strongest immunoreactivity of tumour cells was found at the border of necrotic areas. Our study demonstrates that of all tested components of the matrix metalloproteinase system, only expression of activated MMP-2 correlates with increased malignancy in our melanoma xenograft model, corroborating an important role of MMP-2 in human melanoma invasion and metastasis. © 1999 Cancer Research Campaign

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The bioactivity of matrix metalloproteinases was studied in tissues from colorectal cancer patients by means of both quantitative gelatin zymography and a fluorometric activity assay. Next to paired samples of tumour tissue and distant normal mucosa (n=73), transitional tissue was analysed from a limited (n=33) number of patients. Broad-spectrum matrix metalloproteinase activity and both the active and latent forms of the gelatinases matrix metalloproteinase-2 and -9 were higher in tumour than in normal mucosa. The ratio's between active and latent forms of matrix metalloproteinase-2 and -9 were highest in tumour tissue and normal mucosa, respectively. Matrix metalloproteinase-2 levels, both active and latent forms, correlated inversely with stage of disease, the tumours without synchronous distant metastases containing significantly (P=0.005) more active matrix metalloproteinase-2 than the others. At much lower levels of activity, the same trend was observed in distant normal mucosa. The level of latent form of matrix metalloproteinase-9 in tumour depended on tumour location. Neither the active form of matrix metalloproteinase-9 nor broad-spectrum matrix metalloproteinase activity in tumour tissue did correlate with any of the clinicopathological parameters investigated. The results demonstrate explicit differences between the activity of matrix metalloproteinase-2 and -9, indicating different roles for both gelatinases in tumour progression. Such data are necessary in order to develop rational anti-cancer therapies based on inhibition of specific matrix metalloproteinases.

Tumor cell invasion and metastasis formation depend on both adhesive and proteolytic mechanisms. Previous studies have shown that expression of matrix metalloproteinase-2 and integrin alphavbeta3 correlate with melanoma progression. Recently, direct binding of matrix metalloproteinase-2 to alpha(v)beta3 was implicated in presenting activated matrix metalloproteinase-2 on the cell surface of invasive cells. In this study we investigated this, using the highly metastatic, alpha(v)beta3-negative melanoma cell lines MV3 and BLM, their beta3-transfected alpha(v)beta3 expressing counterparts, xenografts derived from these cell lines, and fresh human cutaneous melanoma lesions comprising all stages of melanoma progression. Expression and activation status of matrix metalloproteinase-2 were studied by reverse transcription-polymerase chain reaction, immunohistochemistry, western blotting, and zymographic analysis, respectively. Matrix metalloproteinase-2 protein expression in vitro was similar in both alpha(v)beta3-negative and alpha(v)beta3-positive cell lines Remarkable differences, however, exist in the localization of inactive and active matrix metalloproteinase-2. Soluble active matrix metalloproteinase-2 was detectable only in the conditioned medium of alpha(v)beta3-negative cell lines and undetectable in the alpha(v)beta3-positive cell lines. Conversely, active matrix metalloproteinase-2 was present exclusively on the cell surface of the alpha(v)beta3 expressing transfectants. Western blot analysis of other components that are involved in matrix metalloproteinase-2 activation showed that processing of proMT1-matrix metalloproteinase to the activated form was enhanced in beta3 transfectants, whereas secretion of tissue inhibitor of metalloproteinase-2 was decreased. In vivo, the presence of functionally active matrix metalloproteinase-2 was significantly higher in xenografts derived from the alpha(v)beta3 expressing MV3 and BLM cell lines. In human cutaneous melanoma lesions, neither matrix metalloproteinase-2 nor integrin alpha(v)beta3 is detectable in melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and alphavbeta3-positive tumor cells was clearly increased in primary melanomas, and melanoma metastases. Double staining experiments and confocal laser microscopy demonstrated that the percentage of cells coexpressing matrix metalloproteinase-2 and alpha(v)beta3 increased in advanced primary melanomas and melanoma metastases. In addition, zymography showed that functionally active matrix metalloproteinase-2 was frequently present in melanoma metastases. In these lesions a high proportion of matrix metalloproteinase-2- and alphavbeta3-double-positive melanoma cells were detectable. Our study demonstrates that the presence of activated matrix metalloproteinase-2 correlates with expression of alpha(v)beta3 in human melanoma cells both in vitro and in vivo, and also in fresh human melanoma lesions. These findings strongly suggest that co-ordinated expr...

Low pro-matrix metalloproteinase-2 levels and high tissue inhibitor of metalloproteinase-1 levels correlate with parameters of colorectal cancer disease. These correlations may be used in the search for new markers in colorectal cancer diagnosis and prognosis.

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These data suggest a role for MMPs in colorectal cancer liver metastasis, but indicate different roles for individual MMPs.