Chemokine-enhanced chemotaxis of lymphangioleiomyomatosis cells with mutations in the tumor suppressor TSC2 gene.

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction caused by LAM cells (smooth-muscle-like cells) that have mutations in the tumor suppressor genes tuberous sclerosis complex (TSC) 1 or 2 and have the capacity to metastasize. Since chemokines and their receptors function in chemotaxis of metastatic cells, we hypothesized that LAM cells may be recruited by chemokine(s) in the lung. Quantification of 25 chemokines in bronchoalveolar lavage fluid from LAM patients and healthy volunteers revealed that concentrations of CCL2, CXCL1, and CXCL5 were significantly higher in samples from LAM patients than those from healthy volunteers. In vitro, CCL2 or MCP-1 induced selective migration of cells, showing loss of heterozygosity of TSC2 from a heterogeneous population of cells grown from explanted LAM lungs. Additionally, the frequencies of single-nucleotide polymorphisms in the CCL2 gene promoter region differed significantly in LAM patients and healthy volunteers (p = 0.018), and one polymorphism was associated significantly more frequently with the decline of lung function. The presence (i.e., potential functionality) of chemokine receptors was evaluated using immunohistochemistry in lung sections from 30 LAM patients. Expression of chemokines and these receptors varied among LAM patients and differed from that seen in some cancers (e.g., breast cancer and melanoma cells). These observations are consistent with the notion that chemokines such as CCL2 may serve to determine mobility and specify the site of metastasis of the LAM cell.

Transforming growth factor (TGF)-beta1-producing regulatory T cells induce Smad-mediated interleukin 10 secretion that facilitates coordinated immunoregulatory activity and amelioration of TGF-beta1-mediated fibrosis.

Interleukin (IL)-10 and transforming growth factor (TGF)-beta1 are suppressor cytokines that frequently occur together during a regulatory T cell response. Here we used a one gene doxycycline (Dox)-inducible plasmid encoding TGF-beta1 to analyze this association and test its utility. In initial studies, we showed that intranasal administration of this plasmid (along with Dox) led to the appearance of TGF-beta1-producing cells (in spleen and lamina propria) and the almost concomitant appearance of IL-10-producing cells. Moreover, we showed that these cells exert Dox-regulated suppression of the T helper cell (Th)1-mediated inflammation in trinitrobenzene sulfonic acid colitis. In subsequent in vitro studies using retroviral TGF-beta1 expression, we established that IL-10 production by Th1 cells occurs after exposure to TGF-beta1 from either an endogenous or exogenous source. In addition, using a self-inactivating retrovirus luciferase reporter construct we showed that TGF-beta1 induces Smad4, which then binds to and activates the IL-10 promoter. Furthermore, intranasal TGF-beta1 plasmid administration ameliorates bleomycin-induced fibrosis in wild-type but not IL-10-deficient mice, strongly suggesting that the amelioration is IL-10 dependent and that IL-10 protects mice from TGF-beta1-mediated fibrosis. Taken together, these findings suggest that the induction of IL-10 by TGF-beta1 is not fortuitous, but instead fulfills important requirements of TGF-beta1 function after its secretion by regulatory T cells.

Genetic polymorphism in collagen type 1 alpha 2 intron 33 can be used for the initial screening of East Asians quickly and easily by a heteroduplex analysis.

To save time and simplify the matching genetic information of bereaved family with the collected samples for identification of individuals in a mass-scale disaster and terrorism using molecular biological methods, we propose a method to initially screen samples according to different populations. We found a unique deletion (26822-3) in an allele that 62.0% of East Asian individuals had in intron 33 of type I collagen alpha 2 (COL1A2), while most Europeans and Africans did not have it. By making a heteroduplex complex, a visual discrimination was possible using 10% PAGE. A PCR and heteroduplex analysis could identify genetic polymorphism of COL1A2 intron 33 both clearly and accurately, even from decomposed samples or very small amounts of samples within a very short time period (approximately within 150min). As a result, genetic polymorphism of COL1A2 intron 33 is thus considered to be useful to quickly and easily screen East Asians from vast number of samples using a heteroduplex analysis. Consequently, we consider that an initial screening method for East Asians which use a heteroduplex analysis of genetic polymorphism of COL1A2 intron 33 can smoothly and effectively perform various identifications among East Asians when supplies are logistically poor after a mass-scale disaster or terrorism.

Expression of mRNAs for telomeric repeat binding factor (TRF)-1 and TRF2 in atypical adenomatous hyperplasia and adenocarcinoma of the lung.

PURPOSE AND EXPERIMENTAL DESIGN: It has been suggested that atypical adenomatous hyperplasia (AAH) may be a precursor of peripheral adenocarcinoma of the lung. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA onto chromosomal ends. Its activity is thought to participate in the development of most human cancers. Telomere-specific DNA-binding proteins, such as telomeric repeat binding factor 1 and telomeric repeat binding factor 2, also control telomere length in a complex interplay with telomerase. Here we investigated the expressions of the mRNAs encoded by the TERF1 and TERF2 genes using in situ hybridization in surgically resected specimens [28 AAHs (11 lesions were interpreted as low-grade AAH, and 17 were interpreted as high-grade AAH) and 40 peripherally located bronchioloalveolar carcinoma (BAC). RESULTS: A clear overexpression of these mRNAs was recognized in low- and high-grade AAH and BAC samples (as compared with normal tissues) using in situ hybridization and these mRNAs were detected in normal AAH and BAC samples using reverse transcription-PCR. The expressions of TERF1 and TERF2 mRNA detected by in situ hybridization were scored positive in 36% and 82% of low-grade AAH, 65% and 83% of high-grade AAH, and 88% and 88% of BAC, respectively. Statistically significant differences in TERF1 mRNA expression could be shown between low-grade AAH and BAC and between high-grade AAH and BAC. There was no statistical difference in the positive expressions of TERF2 mRNA among low-grade AAH, high-grade AAH, and BAC. CONCLUSIONS: These results are consistent with (but are not enough to confirm) the idea that high-grade AAH is closely related to BAC.

Expression of telomerase reverse transcriptase (TERT) in malignant mesotheliomas.

To evaluate the usefulness of determinations of telomerase activity for distinguishing malignant from benign mesothelial lesions, immunohistochemical (using a rabbit polyclonal antibody and the peroxidase method; n = 68) and in situ hybridization (using sense and antisense oligonucleotide probes; n = 46) studies were made on malignant mesotheliomas (epithelioid, 39; sarcomatoid, 18, including 2 of the desmoplastic type; and biphasic, 11) and 19 benign mesothelial lesions (benign mesothelial hyperplasia, 3; and reactive pleuritis, 16). In addition, biochemical studies of telomerase activity were made in 9 of the malignant mesotheliomas. Telomerase activity was detected histochemically in all but one of the malignant mesotheliomas, but only in one (pleuritis) of the benign lesions, in which it was present only in activated lymphocytes. Antisense hybridization signals indicated the presence of telomerase mRNA mainly in the cytoplasm of the malignant cells. Sense probes gave negative results. Biochemical determinations revealed a strong telomerase activity in the 9 malignant mesotheliomas examined. This study demonstrates the usefulness of immunohistochemical staining for the evaluation of mesotheliomas. The required immunostaining can be performed using paraffin sections of formalin-fixed tissues.

Expression of human telomerase RNA component and telomerase reverse transcriptase mRNA in atypical adenomatous hyperplasia of the lung.

It has been suggested that atypical adenomatous hyperplasia (AAH) may be a precursor of peripheral adenocarcinoma of the lung. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA onto chromosomal ends. Its activity is believed to be crucial for cellular immortality, and it is thought to participate in the development of most human cancers. However, little is known about the regulation of telomerase in AAH. We investigated the expression of human telomerase RNA component (hTERC) and telomerase reverse transcriptase (hTERT) mRNA using in situ hybridization in surgically resected formalin-fixed, paraffin-embedded specimens: 29 AAHs (with 11 lesions interpreted as low-grade AAH and 18 as high-grade AAH) and 40 peripherally located nonmucinous bronchioloalveolar carcinomas (NMBACs) measuring < or = 20 mm in greatest diameter. Positive expressions of hTERC and hTERT mRNA were recognized in 27.3% and 27.3% of low-grade AAHs, 72.2% and 77.8% of high-grade AAHs, and 97.5% and 97.5% of NMBACs, respectively. Statistically, significant differences in both expressions could be shown between low-grade AAH and high-grade AAH and between high-grade AAH and NMBAC. Thus, in terms of the incidence of these expressions, high-grade AAH was intermediate between low-grade AAH and NMBAC and tended to be closer to NMBAC. These results suggest that high-grade AAH may be a precursor lesion of peripherally located NMBAC.

Expression of membrane-type-1-matrix metalloproteinase and metalloproteinase-2 in nonsmall cell lung carcinomas.

For the metastasis and invasion of cancer cells, destruction of extracellular matrix is essential. In this process, collagen is broken down by some matrix metalloproteinases. Matrix metalloproteinase 2 (MMP2) is able to cleave type IV collagen, and membrane-type-1-matrix metalloproteinase (MT1-MMP) induces activation of proMMP2. We investigated the expressions of MT1-MMP and MMP2 and their relation to both clinicopathologic parameters and clinical outcome in non-small cell lung carcinomas (NSCLC). Eighty-nine specimens of NSCLC were examined using in situ hybridization and immunohistochemistry. Each metalloproteinase was expressed within the cytoplasm of tumor cells with or without stromal cells in NSCLC. Tumors in which tumor cells strongly stained for MT1-MMP mRNA or protein made up more than 50% of the tumor area were found in 44 and 26% of cases, respectively. The corresponding values for MMP-2 mRNA and protein, were 51 and 26%. Our analysis of clinicopathological findings revealed a significant positive relationship between MT1-MMP mRNA and p-M. The correlation between MMP2 protein-staining status and overall survival rate reached significance in the univariate analysis. However, an association was not demonstrated in the multivariate analysis. The detection of MT1-MMP and MMP2 is likely to be of limited value in informing the prognosis in NSCLC.

Survivin expression in atypical adenomatous hyperplasia of the lung.

Few studies have investigated apoptosis-related factors in atypical adenomatous hyperplasia (AAH) and nonmucinous bronchioloalveolar carcinoma. We studied the expression of survivin, bcl-2, and p53 in 29 AAH lesions (low-grade, 11; high-grade, 18) and 40 nonmucinous BACs using immunohistochemical analysis and of survivin messenger RNA in 6 nonmucinous BACs using reverse transcription-polymerase chain reaction (RT-PCR). The incidence of positive survivin expression was 9% (1/11) in low-grade AAH, 89% (16/18) in high-grade AAH, and 100% (40/40) in nonmucinous BAC. Statistically significant differences were found between low-grade and high-grade AAH and between high-grade AAH and nonmucinous BAC. The percentages obtained for positive bcl-2 and p53 expression were 18% (2/11) and 0% (0/11) in low-grade AAH, respectively, and 28% (5/18) for both in high-grade AAH and 48% (19/40) for both in nonmucinous BAC. In RT-PCR, the intensity of survivin messenger RNA expression was stronger in nonmucinous BACs than in normal lung tissue samples. Thus, the expression of the 3 antibodies in high-grade AAH was intermediate between low-grade AAH and nonmucinous BAC. High-grade AAH may be closely related to nonmucinous BAC.

Mre11 expression in atypical adenomatous hyperplasia and adenocarcinoma of the lung.

CONTEXT: The Mre11-Rad50-NBS1 complex plays an important role in telomere maintenance. Recently, it has been proposed that alterations in Mre11 function may be contributing factors in the development of some tumors. Moreover, mutations of Mre11 have been demonstrated to cause reduced Mre11 immunostaining. OBJECTIVE: To investigate Mre11 in atypical adenomatous hyperplasia (AAH) and nonmucinous bronchioloalveolar carcinoma (NMBAC), an issue not previously explored. DESIGN: We examined (1) the expression of Mre11 protein in 27 AAHs (9 lesions interpreted as low-grade AAH and 18 as high-grade AAH) and 40 NMBACs (using immunohistochemistry) and (2) Mre11 mRNA expression in 1 high-grade AAH and 6 NMBACs (using reverse transcription polymerase chain reaction). For the analysis of immunoreactivity, the intensity and extent of staining were each scored from 0 to 3. These 2 scores were summed to give in each case a final score of 0 to 6. RESULTS: Scores for Mre11 expression were 5.0 +/- 2.1 for low-grade AAH, 5.4 +/- 1.2 for high-grade AAH, and 5.5 +/- 0.9 for NMBAC, and there was no statistically significant difference among these 3 types of lesions. In the reverse transcription polymerase chain reaction for Mre11 mRNA, polymerase chain reaction products were detected in all samples. CONCLUSIONS: On this basis, we suggest that the part played by Mre11 in telomere maintenance may not be important for the progression of the adenoma-carcinoma (AAH-NMBAC) sequence in the lung, although some role for it in carcinogenesis cannot be completely ruled out.

Expression of human telomerase reverse transcriptase in lymphangioleiomyomatosis.

Telomerase synthesizes nucleotide hexameric repeats (telomeres) at the ends of chromosomes, replacing base sequences that are lost from these sites during each mitotic cycle and protecting these ends against the action of exonucleases and ligases. Therefore, telomerase is essential for maintaining cellular replication. To evaluate the role of telomerase in the proliferation of abnormal smooth muscle cells (lymphangioleiomyomatosis [LAM] cells) in LAM, we performed immunostaining and in situ hybridization studies to identify telomerase protein and messenger RNA (mRNA), respectively, in pulmonary (n = 18) and extrapulmonary (n = 4) lesions from 22 women with LAM (14 untreated and 8 treated with progesterone or tamoxifen). Immunoreactivity and hybridization signals for telomerase were observed in 5 to 20% of LAM cells, mostly of the spindle-shaped type, in 21 of the 22 patients, and were less intense in the treated group. Other types of cells were unreactive in both groups. Telomerase colocalized in the same cells with alpha-smooth muscle actin, but only rarely with HMB-45 antibody (a marker for epithelioid LAM cells); colocalization with proliferating cell nuclear antigen was incomplete. The telomerase-positive LAM cells may constitute the sources of renewal of LAM cells. Modulation of telomerase may be involved in the control of LAM cell proliferation.

Molecular and genetic analysis of disseminated neoplastic cells in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a multisystem disorder of women, characterized by cystic degeneration of the lungs, renal angiomyolipomas (AML), and lymphatic abnormalities. LAM lesions result from the proliferation of benign-appearing, smooth muscle-like LAM cells, which are characterized by loss of heterozygosity (LOH) of one of the tuberous sclerosis complex (TSC) genes. LAM cells are believed to migrate among the involved organs. Because of the apparently metastatic behavior of LAM, we tried to isolate LAM cells from body fluids. A cell fraction separated by density gradient centrifugation from blood had TSC2 LOH in 33 of 60 (55%) LAM patients. Cells with TSC2 LOH were also found in urine from 11 of 14 (79%) patients with AML and in chylous fluid from 1 of 3 (33%) patients. Identification of LAM cells with TSC2 LOH in body fluids was not correlated with severity of lung disease or extrapulmonary involvement and was found in one patient after double lung transplantation. These studies are compatible with a multisite origin for LAM cells. They establish the existence of disseminated, potentially metastatic LAM cells through a relatively simple, noninvasive procedure that should be valuable for molecular and genetic studies of somatic mutations in LAM and perhaps other metastatic diseases.