Paralemnin of the lens

Paralemmin was identified in the chicken lens as a protein with mol. wt 65 kDa and a splice variant of 60 kDa, both soluble in Triton X-100. Paralemmin is localized to the plasma membrane of fiber cells, and was not detected in the annular pad cells. Thus in the chick lens it is another feature of fiber cell differentiation. Its localization to the short side of the fiber cell and the sites of fiber cell interlocking suggests that paralemmin may play a role in the development of such interdigitating processes.     

Promoter Hypermethylation of ARID1A Gene Is Responsible for Its Low mRNA Expression in Many Invasive Breast Cancers

ARID1A (AT-rich interactive domain 1A) has recently been identified as a tumor suppressor gene. Its mRNA expression is significantly low in many breast cancers; this is often associated with more aggressive phenotypes. However, the underlying molecular mechanism for its low expression has not been fully understood. This study was undertaken to evaluate the contribution of gene copy number variation, mutations, promoter methylation and histone modification to ARID1A’s low expression. 38 pairs of breast invasive ductal carcinomas and their normal breast tissue counterparts from the same patients were randomly selected for gene expression and copy number variation detection. Promoter methylation and histone modification levels were evaluated by MeDIP-qPCR and ChIP-qPCR, respectively. PCR product Sanger sequencing was carried out to detect the exon mutation rate. Twenty-two out of 38 invasive ductal carcinomas in the study (57.9%) revealed ARID1A mRNA low expression by realtime RT-PCR. The relative promoter methylation level was, significantly higher in ARID1A mRNA low expression group compared with its high expression group (p<0.001). In the low expression group, nineteen out of 22 invasive ductal carcinomas (86.4%) exhibited ARID1A promoter hypermthylation. In addition, the promoter hypermethylation was accompanied with repressive histone modification (H3K27Me3). Although five out of 38 invasive ductal carcinomas (13.2%) exhibited loss of ARID1A gene copy number by realtime PCR and nine exon novel mutations are seen from eight out of 33 invasive ductal carcinomas (24.2%), there was no statistically significant difference in both ARID1A mRNA low and high expression groups (p = 0.25,and p = 0.68, respectively). We demonstrate that promoter hypermethylation was the main culprit for ARID1A mRNA low expression in invasive ductal carcinomas. The influence of mutation and copy number variation on the expression were statistically insignificant at mRNA level, and were, therefore, not considered the main causes for ARID1A mRNA low expression in invasive breast cancer.     

Estrogen receptor beta splice variant mRNAs are differentially altered during breast carcinogenesis

We previously identified 10 exon deletion ERbeta variant mRNAs in various human tissues [FEBS Lett. 516 (2002) 133]. In the current study, we have investigated the expression of these variant mRNAs in normal breast tissues and their alterations in cancer tissues. A total of 43 cancer tissues in comparison with their matched normal tissues were analyzed by RT PCR using the newly developed 'Splice Targeted Primer Approach'. The data presented here show that normal breast tissues express 9 of the 10 identified variant mRNAs. Of the nine variants, the mRNAs with exons 5-6 deletions were significantly decreased ( approximately 80%) in a large majority of cancer tissues (two-sided paired t-test, n=43 patients, P<0.00001). The expression of ERbeta exon 5Delta, that could potentially have transactivating property in the absence of hormone, was changed differently among different grade tumors (analysis of variance F-test, n=43 patients, P=0.0452; Kruskal-Wallis test, n=43 patients, P=0.0356). When change in expression of ERbeta exon 5Delta mRNA levels was used as a categorical variable, a significant association was found between the change status (increase, no change, decrease) of this variant and grade of the tumor (Fischer's exact test, n=43 patients, P=0.0129). In particular, it was significantly increased in grade III tumors and decreased in grade II tumors. This variant was also changed differently in pre- and post-menopausal women. Its expression levels were increased in the tumors of post-menopausal women (mean change=3.6685), while they were decreased in pre-menopausal women (mean change=-24.3662). Thus a significant association was observed between the expression of this variant and menopausal status (a two-sided paired t-test, n=43 patients, P=0.03). Other variants were either expressed at very low frequency or not significantly altered.     

Arylamine N-acetyltransferase-1 is highly expressed in breast cancers and conveys enhanced growth and resistance to etoposide in vitro

Comparative two-dimensional proteome analysis was used to identify proteins differentially expressed in multiple clinical normal and breast cancer tissues. One protein, the expression of which was elevated in invasive ductal and lobular breast carcinomas when compared with normal breast tissue, was arylamine N-acetyltransferase-1 (NAT-1), a Phase II drug-metabolizing enzyme. NAT-1 overexpression in clinical breast cancers was confirmed at the mRNA level and immunohistochemical analysis of NAT-1 in 108 breast cancer donors demonstrated a strong association of NAT-1 staining with estrogen receptor-positive tumors. Analysis of the effect of active NAT-1 overexpression in a normal luminal epithelial-derived cell line demonstrated enhanced growth properties and etoposide resistance relative to control cells. Thus, NAT-1 may not only play a role in the development of cancers through enhanced mutagenesis but may also contribute to the resistance of some cancers to cytotoxic drugs.     

Identification and characterization of a novel alternative splice variant of mouse GMx33alpha/GPP34

We have cloned and characterized a novel splice variant of mouse GMx33alpha/Golgi-associated protein of 34 kDa (GPP34), hereby designated GMx33alphaV/GPP34V. This splice variant skips the second and third exons, and the resulting frame shift generates a stop codon in the fourth exon. GMx33alphaV/GPP34V is comprised of 81 amino acid residues derived from the N-terminal end of the full length protein and corresponds to approximately one-third of the full length GMx33alpha/GPP34 sequence with a calculated molecular mass of 8900. In contrast to GMx33alpha/GPP34 mRNA which is expressed at similar levels in various tissues, GMx33alphaV/GPP34V mRNA was differentially expressed when examined by RT-PCR. Compared to other tissues, skeletal muscle showed relatively strong expression of GMx33alphaV/GPP34V mRNA. This splice variant cDNA was also detected in a human cell line.     

Novel immunohistochemical markers for the differentiation of lobular and ductal invasive breast carcinomas

AIMS. Invasive ductal and lobular carcinomas are the most common histological types of breast cancer. The loss of E-cadherin expression has been suggested to be the most reliable marker for invasive lobular carcinoma. The aim of our study was to identify the diagnostic usefulness of novel markers in the differentiation of these tumor types. METHODS. We examined tissue microarrays (TMA) which were constructed from surgical specimens of 119 breast cancer patients. TMA consisted of 80 ductal carcinomas, 29 lobular carcinomas and special type cancers. TMA sections were stained using standard immunohistochemical methods. Monoclonal mouse antibodies against E-cadherin, cytokeratin 5/6 and 17, and polyclonal mouse antibodies against EMP1, DDR1, PRKCI and DVL1 were used. RESULTS. E-cadherin was absent in 93.3% of lobular tumors compared with only 15 % of ductal tumors (p<0.0001). EMP1 and DVL1 were overexpressed in lobular tumors (93.1% and 96.5%, respectively), whereas PRKCI and DDR1 were positive in ductal cancers (90% and 96.2%, respectively). Reduced expression or absence of both cytokeratins 5/6 and 17 was found in both tumor tissues in comparison to normal terminal duct lobular units (p<0.0001). CONCLUSIONS. Apart from the well-established marker, E-cadherin, proteins examined on TMA slides by immunohistochemistry (EMP1, DVL1, DDR1, PRKCI) may represent novel tissue markers helpful in the differentiation of ductal and lobular breast cancers. Further studies with larger sets of patients are desirable, to verify the complete immunohistochemical profiles of various histological types of breast cancer and determine the prognostic and predictive significance of novel markers.     

A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps

A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors has been shown to confer metastatic potential to a non-metastasizing rat pancreatic carcinoma cell line and to non- metastasizing sarcoma cells. Homologues of this variant as well as several other CD44 splice variants are also expressed at the RNA level in human carcinoma cell lines from lung, breast, and colon, and in immortalized keratinocytes. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we studied the expression of variant CD44 glycoproteins in normal human tissues and in colorectal neoplasia. Expression of CD44 variant proteins in normal human tissues was readily found on several epithelial tissues including the squamous epithelia of the epidermis, tonsils, and pharynx, and the glandular epithelium of the pancreatic ducts, but was largely absent from other epithelia and from most non-epithelial cells and tissues. In human colorectal neoplasia CD44 variant proteins, including homologues of those which confer metastatic ability to rat tumors, were found on all invasive carcinomas and carcinoma metastases. Interestingly, focal expression was also observed in adenomatous polyps, expression being related to areas of dysplasia. The distribution of the CD44 variants in human tissues suggests that they play a role in a few restricted differentiation pathways and that in colorectal tumors one of these pathways has been reactivated. The finding that metastasis-related variants are already expressed at a relatively early stage in colorectal carcinogenesis and tumor progression, i.e., in adenomatous polyps, suggests the existence of a yet unknown selective advantage linked to CD44 variant expression. The continued expression in metastases would be compatible with a role in the metastatic process.     

The Expression of the Zonula Adhaerens Protein PLEKHA7 Is Strongly Decreased in High Grade Ductal and Lobular Breast Carcinomas

PLEKHA7 is a junctional protein, which participates in a complex that stabilizes E-cadherin at the zonula adhaerens. Since E-cadherin is involved in epithelial morphogenesis, signaling, and tumor progression, we explored PLEKHA7 expression in cancer. PLEKHA7 expression was assessed in invasive ductal and lobular carcinomas of the breast by immunohistochemistry, immunofluorescence and quantitative RT-PCR. PLEKHA7 was detected at epithelial junctions of normal mammary ducts and lobules, and of tubular and micropapillary structures within G1 and G2 ductal carcinomas. At these junctions, the localization of PLEKHA7 was along the circumferential belt (zonula adhaerens), and only partially overlapping with that of E-cadherin, p120ctn and ZO-1, as shown previously in rodent tissues. PLEKHA7 immunolabeling was strongly decreased in G3 ductal carcinomas and undetectable in lobular carcinomas. PLEKHA7 mRNA was detected in both ductal and lobular carcinomas, with no observed correlation between mRNA levels and tumor type or grade. In summary, PLEKHA7 is a junctional marker of epithelial cells within tubular structures both in normal breast tissue and ductal carcinomas, and since PLEKHA7 protein but not mRNA expression is strongly decreased or lost in high grade ductal carcinomas and in lobular carcinomas, loss of PLEKHA7 is a newly characterized feature of these carcinomas.     

Expression of the apoptosis-inducing ligands FasL and TRAIL in malignant and benign human breast tumors

Apoptosis-inducing ligands such as Fas ligand (FasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have been found to play an important role in cell regulation. Different malignant tumors show an altered expression of these ligands and their respective receptors compared to normal tissues. The purpose of this study was therefore to investigate expression of TRAIL, FasL, and its receptor Fas on protein and mRNA levels in breast carcinomas (n=40), fibroadenomas (n=7), and normal breast tissues (n=5). Immunohistochemical reaction demonstrated that FasL was strongly expressed in breast cancer tissues (34/40) while only one fibroadenoma and one normal breast tissue reacted weakly positive for FasL. All fibroadenomas and normal breast tissues as well as the majority of breast cancer tissues expressed Fas on protein level. Quantitative RT-PCR analysis detected high expression of FasL mRNA in breast cancer tissues and fibroadenomas, whereas fibroadenomas showed the highest Fas mRNA copy numbers, followed by breast cancer tissues and normal breast tissues (P<0.05). Compared to FasL expression, TRAIL could be detected in less breast cancer tissues on protein level (21/40) and was found in only one fibroadenoma and none of the normal breast tissues. Thus, it can be concluded that malignant breast tumors show an altered expression of the two apoptosis-inducing ligands FasL and TRAIL. Accepted: 4 January 2000     

Splice variants of human cathepsin L mRNA show different expression rates.

Human cathepsin L (hCATL) mRNA occurs in vivo in at least three splice variants. They differ in the length of exon 1, which comprises 278 nucleotides (hCATL-A), 188 nucleotides (hCATL-A2) and 132 nucleotides (hCATL-A3), respectively. We describe here the shortest variant for the first time. This form is predominant in all tissues and cells examined so far, including malignant tumors. We studied the expression rate of the three mRNA variants in order to explain why malignant kidney tumors show low cathepsin L activity despite of high mRNA levels. The variant hCATL-A3 showed the highest expression rate in vitro and in vivo. Based on these results, we suggest a cis-acting element on human cathepsin L mRNA which can be bound by a negative trans-acting regulator, thus leading to reduced expression rates.