[Perivascular epithelioid cell tumor of the lung: a clinicopathological analysis of eight cases].

Objective: To investigate the clinicopathological features of perivascular epithelioid cell tumor (PEComa) of the lung. Methods: Eight PEComa cases of the lung diagnosed at the First Affiliated Hospital of Soochow University, Suzhou, China from July 2008 to December 2021 were collected and subject to immunohistochemical staining, fluorescence in situ hybridization and next generation sequencing. The relevant literature was reviewed and the clinicopathological features were analyzed. Results: There were 5 males and 3 females, aged from 18 to 70 years (mean 39 years). There were 3 cases of the right upper lung, 3 cases of the left lower lung, 1 case of the left upper lung and 1 case of the right middle lung. Seven cases were solitary and 1 case was multifocal (4 lesions). Seven cases were benign while one was malignant. The tumors were all located in the peripheral part of the lung, with a maximum diameter of 0.2-4.0 cm. Grossly, they were oval and well circumscribed. Microscopically, the tumor cells were oval, short spindle-shaped, arranged in solid nests, acinar or hemangiopericytoma-like patterns, with clear or eosinophilic cytoplasm. The stroma was rich in blood vessels with hyalinization. Coagulated necrosis and high-grade nuclei were seen in the malignant case, and calcification was seen in 2 cases. Immunohistochemically, the tumor cells were positive for Melan A (8/8), HMB45 (7/8), CD34 (6/8), TFE3 (4/7), and SMA (3/8). All cases were negative for CKpan and S-100. TFE3 (Xp11.2) gene fusion was examined using the TFE3 break-apart fluorescence in situ hybridization in 5 cases, in which only the malignant case was positive. The next generation sequencing revealed the SFPQ-TFE3 [t(X;1)(p11.2;p34)] fusion. Follow-up of the patients ranged from 12 to 173 months while one patient was lost to the follow-up. The malignant case had tumor metastasis to the brain 4 years after the operation and then received radiotherapy. Other 6 cases had no recurrence and metastasis, and all the 7 patients survived. Conclusions: Most of the PEComas of the lung are benign. When there are malignant morphological features such as necrosis, high-grade nuclei or SFPQ-TFE3 gene fusion, close follow-up seems necessary.

Enhanced apoptosis under low serum conditions in human glioblastoma cells by connexin 43 (Cx43).

Connexin 43 (Cx43), a structural component of gap junctions, is believed to function as a tumor suppressor gene. Previously, we showed that expression of Cx43 suppresses cell proliferation and tumorigenicity of human glioblastoma cells [Huang et al., Cancer Res 58:5089-5096, 1998] and enhances apoptosis in response to chemotherapeutic agents [Huang et al., Int J Cancer 92:130-138, 2001]. In the present study, we demonstrated that expression of Cx43 in human glioblastoma cells potentiated an apoptotic program under low-serum conditions. The Cx43-mediated effect was coupled with a decreased expression of the specific apoptosis-inhibitor bcl-2. Overexpression of bcl-2 in Cx43-transfected cells conferred resistance to apoptosis induced under low-serum conditions, suggesting that the Cx43-mediated apoptosis under low-serum conditions is regulated, in part, through the downregulation of bcl-2 expression. Furthermore, application of the phosphatidylinositol-3'-OH kinase inhibitor LY294002 specifically induced apoptosis in Cx43-transfected cells. Our results demonstrate a new role of Cx43 in the mediation of apoptosis under low serum conditions.

Hydrogen peroxide promotes transformation of rat liver non-neoplastic epithelial cells through activation of epidermal growth factor receptor.

Previous studies demonstrated that hydrogen peroxide (H(2)O(2)) is a tumor promoter in the rat liver epithelial cell line T51B. We investigated the pathway linking H(2)O(2) to tumor promotion. H(2)O(2) can directly induce tyrosine phosphorylation of epidermal growth factor receptor (EGFR). H(2)O(2) and epidermal growth factor exerted similar effects on the induction of early growth response genes, disruption of gap junction communication, triggering of calcium inflow, and promotion of transformation. Furthermore, the effect of H(2)O(2) on tumor promotion was blocked by abrogation of EGFR activation. Our results suggested that tumor promotion by H(2)O(2) is mediated mainly through activation of EGFR in T51B cells.

Connexin 43 (cx43) enhances chemotherapy-induced apoptosis in human glioblastoma cells.

Stable re-expression of connexin 43 (cx43) in human glioblastoma suppresses transformation and tumorigenicity. The present study was designed to examine the role of cx43 in chemotherapy-induced apoptosis. Expression of cx43 in human glioblastoma cells significantly increased sensitivity to several common chemotherapeutic agents, including etoposide, paclitaxel (Taxol) and doxorubicin, compared with control-transfected cells. The increased sensitivity to chemotherapeutic agents resulted from apoptosis as evidenced by Hoechst dye staining, TUNEL assay and annexin V assay. These cx43-mediated effects were coupled with decreased expression of the specific apoptosis inhibitor bcl-2. Over-expression of bcl-2 in cx43-transfected cells partially confers the resistance to apoptosis induced by etoposide, suggesting that the cx43-mediated apoptosis to chemotherapeutic agents is regulated in part through the down-regulation of bcl-2 expression. Furthermore, the cx43-mediated apoptosis in response to chemotherapeutic drugs may not be linked to increased gap junctional communication in cx43-transfected cells. Our results demonstrate a new role of cx43 in the mediation of apoptosis during chemotherapy.

p53 and Egr-1 additively suppress transformed growth in HT1080 cells but Egr-1 counteracts p53-dependent apoptosis.

The human fibrosarcoma cell line, HT1080, clone H4, was used to determine if the transformation suppressive functions of p53 and Egr-1 have the same underlying mechanism. This cell line expresses only mutant p53 and no detectable Egr-1. H4 clones stably expressing Egr-1 are less transformed in proportion to the level of Egr-1 expressed, acting through the induction of the TGFbeta1 gene. Here, H4 cells and the highest Egr-1 expressing clone were transfected with a vector expressing normal human p53 to derive stable clones expressing p53. The expression of p53 in H4 cells inhibited transformed growth and reduced tumorigenicity. The effect of coexpression of both p53 and Egr-1 was additive, producing cell lines with 30% of normal growth rate and sevenfold reduced tumorigenicity compared with control lines. These results indicated that each factor may act independently by different pathways, although each additively increased the level of p21WAF1 cell cycle inhibitor. However, exposure of the H4-derived cells to UV-C irradiation produced contrasting effects. Cell cycle analyses showed that the presence of p53 was associated with loss of the G1 and S cells to apoptosis after irradiation. In contrast, the expression of Egr-1 increased entry into S/G2 phase of the cell cycle with little apoptosis via a mechanism involving elevated FAK and low caspase activities. Apoptosis was observed only in the cell lines that expressed no Egr-1, especially those expressing wt-p53, and was preceded by high caspase activity. In summary, Egr-1 suppressed transformation and counteracted apoptosis by the coordinated activation of TGFbeta1, FN, p21 and FAK, leading to enhanced cell attachment and reduced caspase activity. In the doubly expressing cell line, the survival effect of Egr-1 was dominant over the apoptotic effect of p53.

Tumor promotion by hydrogen peroxide in rat liver epithelial cells.

Reactive oxygen species, including H2O2, play an important role in the tumor promotion process. Using an in vitro model of tumor promotion involving the rat liver epithelial oval cell line T51B, the tumor promoting activity of H2O2 in N-methyl-N'-nitro-N-nitrosoguanidine-initiated cells was studied. In this assay system, the promoting effect of H2O2 is evidenced by the formation of colonies in soft agar, appearance of foci in monolayer culture, disruption of gap junction communication (GJC) in foci areas and growth at higher saturation densities. H2O2 preferentially induced the expression of c-fos, c-jun, c-myc and egr-1, while JunB and JunD levels remained almost unchanged. H2O2 also induced hyperphosphorylation of Cx43 and disruption of GJC. The effects of H2O2 on tumor promotion, induction of immediate early (IE) genes and disruption of GJC are blocked by antioxidants. These results suggest that H2O2 acts as a tumor promoter in rat liver non-neoplastic epithelial cells and that the induction of IE genes and disruption of GJC are two possible targets of H2O2 during the tumor promotion process.

The transcription factor EGR-1 suppresses transformation of human fibrosarcoma HT1080 cells by coordinated induction of transforming growth factor-beta1, fibronectin, and plasminogen activator inhibitor-1.

Re-expression of EGR-1 in fibrosarcoma HT1080 suppresses transformation including tumorigenicity (Huang, R.-P., Liu, C., Fan, Y., Mercola, D., and Adamson, E. (1995) Cancer Res. 55, 5054-5062) owing in part to up-regulation of the transforming growth factor (TGF)-beta1 promoter by EGR-1 which suppresses growth by an autocrine mechanism (Liu, C., Adamson, E., and Mercola, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11831-11836). Here we show that enhanced cell attachment contributes to the suppression via increased secretion of fibronectin (FN) and also of plasminogen activator inhibitor-1 (PAI-1). The secretion of FN and PAI-1 is strongly correlated with EGR-1 expression (RPEARSON = 0.971 and 0. 985, respectively). Addition of authentic TGF-beta1 to parental cells greatly stimulated secretion of PAI-1 but not FN, whereas addition of TGF-beta antibody or lipofection with specific antisense TGF-beta1 oligonucleotides to EGR-1-regulated cells completely inhibits the secretion of PAI-1 but not FN. However, in gel mobility shift assays pure EGR-1 or nuclear extracts of EGR-1-regulated cells specifically bind to two GC-rich elements of the human FN promoter at positions -75/-52 and -4/+18, indicating that the increased secretion of FN is likely due to direct up-regulation by EGR-1. Moreover, adhesion was greatly enhanced in EGR-1-regulated cells and was reversed by treatment with Arg-Gly-Asp (RGD) or PAI-1 antibody indicating that the secreted proteins are functional. We conclude that EGR-1 regulates the coordinated expression of gene products important for cell attachment ("oikis" factor) and normal growth control.

Reduced connexin43 expression in high-grade human brain glioma cells.

BACKGROUND AND OBJECTIVES: Connexin43 (cx43), a gap junction protein, is implicated in the suppression of tumor cell growth. Numerous cancer cells show a reduction or loss of cx43 expression compared to their normal counterparts. Our previous studies suggest that cx43 expression is decreased in a variety of human brain tumor cell lines. To further investigate the role of cx43 in the development of human gliomas, we performed the present study on human glioma grades I-IV. METHODS: Immunohistochemistry was performed on paraffin-embedded tissue sections of 18 human gliomas to analyze the expression levels of cx43 in different stages of human gliomas. RESULTS: High levels of cx43 were observed in all normal brain tissue and in glioma grades I and II. In contrast, the expression of cx43 was very weak in grade III gliomas and almost undetectable in grade IV gliomas. CONCLUSIONS: Our data support the hypothesis that reduction of cx43 is involved in the progression of human gliomas.

Impaired expression and posttranslational processing of connexin43 and downregulation of gap junctional communication in neoplastic human prostate cells.

BACKGROUND: Gap junctional communication (GJC) has been implicated in the control of cell proliferation. Numerous cancer cells show a decrease or loss of GJC compared to their normal counterparts. Lack of adequate information on the status of gap junctions during prostate neoplasia prompted us to examine this form of cancer, which comprises about 14% of male cancer deaths in America. METHODS: Cultured normal human prostate epithelial cells and several different human prostate tumor lines were used in this study. GJC was assayed by dye transfer, whereas Western blot and immunofluorescence methods were used to examine connexin43 (Cx43) levels and the presence of gap junctions, respectively. RESULTS: Normal human prostate cultures exhibited extensive cell-communication which was completely absent in all the examined tumor cells. This disrupted communication was associated with a decreased expression and an impaired posttranslational modification of Cx43 in these cells. Abundant immunostaining of gap junctional channels by a Cx43-antibody was observed in normal prostate cells but not in tumor cells. CONCLUSIONS: Our data provide further support for the hypothesis that loss of junctional communication is a critical step in progression to human prostate neoplasia.

Reversion of the neoplastic phenotype of human glioblastoma cells by connexin 43 (cx43).

Connexins (cx), structural components of gap junction, are believed to play a role in the regulation of cell proliferation and suppression of the neoplastic phenotype. We used human brain glioblastoma tumor cells as a model system to test this hypothesis. Western blot and reverse transcription-PCR analysis indicate that the expression levels of the gap junction protein connexin 43 (cx43) are profoundly decreased in several human brain tumor cell lines examined. Transfection of human cx43 into human glioblastoma cell lines U251 and T98G profoundly reduces cell proliferation in monolayer culture, in soft agar, and in athymic nude mice. Surprisingly, these effects are not associated with the establishment of gap junction communication in cx43 transfected cells. We conclude that the loss of cx43 expression may play a role in the development of human gliomas and that cx43 acts as a tumor suppressor gene to human glioblastoma.

Suppression of human fibrosarcoma cell growth by transcription factor, Egr-1, involves down-regulation of Bcl-2.

Previously, we showed that the transcription factor Egr-1 suppressed the proliferation of v-sis transformed NIH3T3 cells and also a number of human tumor cells. Here, we investigate the possible mechanisms responsible for this function. We show that transfected Egr-1 in human fibrosarcoma cells HT1080 leads to down-regulation of Bcl-2. Transient CAT transfection assays reveal that expression of Egr-1 suppresses Bcl-2 promoter activity in a dose-dependent manner. Furthermore, overexpression of Bcl-2 in Egr-1-expressing HT1080 cells enhanced cell proliferation in monolayer culture and increased anchorage-independent growth. Our results suggest that suppression of tumor cell proliferation by Egr-1 may be at least partially mediated through the down-regulation of Bcl-2.

Expression of multiple apoptosis-regulatory genes in human breast cancer cell lines and primary tumors.

The expression of several apoptosis-regulating genes was evaluated in 9 human breast cancer cell lines, 2 immortalized human mammary epithelial lines, 1 normal breast tissue biopsy, and 3 primary breast tumors, using a multiple antigen detection (MAD) immunoblotting method. The anti-apoptotic proteins Bcl-2, Bcl-X(L), Mcl-1, and BAG-1 were present at immunodetectable levels in 7, 10, 10, and 9 of the 11 lines. Comparing these 11 cell lines among themselves revealed that steady-state levels of Bcl-2, Bcl-X(L), Mcl-1, and BAG-1 were present at relatively higher levels in 4, 6, 5, and 5 of the lines, respectively. In contrast, the pro-apoptotic proteins Bax and Bak were detected in all 11 cell lines, and were present at relatively higher levels in 10 and 5 of the 11 lines, respectively. The Interleukin-1beta converting enzyme (ICE) homolog CPP32 (Caspase-3) was expressed in 10/11 breast cell lines. High levels of p53 protein, indicative of mutant p53, were found in 8 of the 11 lines and correlated inversely with Bax expression (p = 0.01). Bcl-2 and BAG-1 protein levels were positively correlated (p = 0.03). Immunoblot analysis of primary adenocarcinomas revealed expression of the anti-apoptotic proteins Bcl-2, Bcl-X(L), Mcl-1, and BAG-1, as well as the pro-apoptotic proteins Bax, Bak, and CPP32, in at least 2 of the 3 tumors examined. Immunohistochemical analysis was also performed for all of these proteins using 20 paraffin-embedded breast cancer biopsy specimens that all contained residual normal mammary epithelium in combination with both invasive cancer and carcinoma in situ. All of these apoptosis-regulating proteins were detected in primary breast cancers, though the percentage of immunopositive tumor cells varied widely in some cases. Comparisons of the intensity of immunostaining in normal mammary epithelium and invasive carcinoma suggested that Bcl-2 immunointensity tends to be lower in cancers than normal breast epithelium (p = 0.03), whereas CPP32 immunointensity was generally higher in invasive cancers (p < 0.0001). Taken together, the results demonstrate expression of multiple apoptosis-modulating proteins in breast cancer cell lines and primary tumors, suggesting complexity in the regulation of apoptosis in these neoplasms of mammary epithelial origin.

Egr-1 inhibits apoptosis during the UV response: correlation of cell survival with Egr-1 phosphorylation.

UV irradiation of normal or immortalized cells induces a rapid increase in the expression of several transcription factors and is thought to serve a protective function. The human fibrosarcoma cell line, HT1080 clone H4, expresses almost undetectable levels of Egr-1 and does not respond to UV-C irradiation by the induction of Egr-1. The H4 cells are hypersensitive to UV which induces apoptosis and reduces clonogenicity. The introduction of exogenous Egr-1 into H4 (H4E9 and H4E4 cell-lines) confers protection from UV damage as measured by a number of assays. In both NIH3T3 (with inducible Egr-1) and H4E9 (constitutive Egr-1) cells, UV irradiation gave enhanced transactivation of Egr-1 reporters that correlated with phosphorylated Egr-1. Studies using inhibitors indicated that protein kinase-C and tyrosine kinases are involved in the anti-apoptotic effects of Egr-1 after UV damage. This is the first description of a biological effect of phosphorylated Egr-1.

Reciprocal modulation between Sp1 and Egr-1.

Many ubiquitously expressed genes, including oncogenes, lack a proximal TATA or CAAT box but have a region of G + C-rich sequences that appears to replace the usual promoter initiation site. The zinc-finger protein Sp1 is one of the prevalent activators of these genes. The Egr-1 zinc-finger protein has a similar binding site and if the two sites occur in the same region, a variety of activation or inhibitory responses may be obtained. We show that competition between the two factors for overlapping sites on growth-promoting genes could explain why the overexpression of Egr-1 suppresses transformed growth in a number of cell types [Huang et al. (1995): Cancer Res 55:5054-5062; Huang et al. (1997): Int J Cancer]. We demonstrate here that Egr-1 and Sp1 can bind to the same G + C-rich sites and that Egr-1 can displace Sp1 and hence inhibit its activity. We measured the responses of synthetic consensus binding sites and natural promoter sequences linked to a reporter gene and showed that Egr-1 inhibited the activation of transcription by Sp1 on overlapping Sp1/Egr-1 sites. In contrast, Sp1 activity could be augmented by Egr-1 at nonoverlapping sites in the Egr-1 gene promoter, in transient reporter gene studies in Drosophila SL2 cells. In addition, over-expression of exogenous Sp1 in mammalian cells, also leads to increased Egr-1 protein expression, which further inhibits Sp1 transactivation of numerous genes. Therefore, we can account for some of the complex responses of G + C-rich enhancer/promoters by a form of "facilitated inhibition" of Sp1 by Egr-1 at overlapping sites.

Decreased Egr-1 expression in human, mouse and rat mammary cells and tissues correlates with tumor formation.

We have examined several types of tumor cell lines and shown that they invariably expressed little or no Egr-1, in contrast to their normal counterparts. We have previously shown that the expression of exogenous Egr-1 in human breast and other tumor cells markedly reduces transformed growth and tumorigenicity. We therefore hypothesized that the loss of Egr-1 expression plays a role in transformation. All human and mouse breast cancer cell lines and tumors examined had reduced Egr-1 expression compared with their normal counterparts. Reduced Egr-1 expression was also observed in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, and this level increased to normal levels in tumors that regressed after tamoxifen treatment. We concluded, therefore, that loss of Egr-1 expression may play a role in the deregulation of normal growth in the tumorigenic process and that Egr-1 acts as a tumor suppressor gene.

UV activates growth factor receptors via reactive oxygen intermediates.

Exposure of mammalian cells to UV irradiation induces rapid and transient expression of early growth response-1 gene (Egr-1) encoding a transcription factor that plays a role in cell survival. These signals from the irradiated cell surface are likely to involve more than one pathway, and we show here that an essential pathway involves activation of several growth factor receptors by reactive oxygen intermediates (ROI). UVC irradiation causes the tyrosine phosphorylation of EGF receptor (EGFR) in mouse NIH 3T3 fibroblasts and HC11 mouse mammary cells. EGFR activation by irradiation of cells is abrogated by suramin, by antioxidants, and by the presence of a dominant negative EGFR. UV induces the formation of complexes between activated EGFR and SOS, Grb2, PLC gamma, and SHC that can be precipitated with antibodies to EGFR. The activation of EGFR by UV is mimicked by H2O2, suggesting that ROI may function upstream of EGFR activation. Our observations support the hypothesis that ROI and growth factor receptors operate in the early steps of the UV signal that lead to the enhanced expression and activity of Egr-1.

Egr-1 negatively regulates human tumor cell growth via the DNA-binding domain.

Human HT1080 fibrosarcoma cells, subclone H4, express little or no Egr-1 (Zif/268, Krox 24), an early growth response gene encoding a transcription factor. Phorbol ester (but not serum) treatment only can elicit a small increase in Egr-1 expression in H4, in contrast to the normally rapid, high transient expression of Egr-1 observed after the addition of a wide range of stimulating agents to normal or immortalized cell lines. Because several human tumor cell lines express little Egr-1, we tested the hypothesis that this loss was causal to transformation. We report here that the expression of exogenous mouse Egr-1 in H4 cells inhibits transformed growth in a dose-dependent manner and significantly suppresses tumorigenicity in athymic mice. By overexpression of the fragment in Egr-1 that is responsible for its DNA-binding activity, the zinc-finger domain, we show that this domain has a similar activity. Moreover, the expression of antisense mRNA encoding the DNA-binding domain increases the transformed character of the H4 cells. One possible conclusion is that endogenous Egr-1-like genes perform growth-regulatory functions. Other human tumor lines are also growth suppressed by Egr-1 overexpression including ZR-75-1 breast carcinoma, U251 glioblastoma, and to a lesser extent, SAOS-2 osteosarcoma cells. These results are surprising in light of the "early growth response" character of Egr-1 but extend our earlier report of suppression of growth in v-sis-transformed NIH3T3 cells.

Detection and location of amphiregulin and Cripto-1 expression in the developing postnatal mouse mammary gland.

Amphiregulin (Ar) and Cripto-1 (Cr-1) are growth promoting peptides that share amino acid sequence homology with epidermal growth factor (EGF). The present study examined Ar and Cr-1 mRNA and protein expression during various stages of C57BL/6 mouse mammary morphogenesis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect transcripts for Ar and Cr-1 at all stages of mammary development. Immunocytochemical (ICC) localization demonstrated that in virgin 4-week to mature 12-week-old mouse fourth inguinal mammary gland, Ar and Cr-1 are expressed in the stromal cells, luminal epithelial cells, and myoepithelial cells of the branching ducts. Ar, and to lesser extent Cr-1, were also found in the epithelial cap cells and in the luminal epithelial cells of the advancing terminal end bud (TEB) from virgin 4-week and 6-week-old mice. Western blot analysis demonstrated that both Ar (28 and 26 kDa) and Cr-1 (90, 67, 56, and 21 kDa) proteins are expressed in virgin, 13.5 day midpregnant and in the 14 day lactating mammary gland. In addition, Ar and Cr-1 are associated with developing alveolar structures as determined by ICC. These results imply that together with EGF and transforming growth factor alpha (TGF alpha), Ar and Cr-1 may play salient roles as modifiers in the morphogenesis and differentiation of the mammary gland.

A biological role for Egr-1 in cell survival following ultra-violet irradiation.

The response to ultra-violet (u.v.) irradiation varies among cells, but commonly involves the rapid increase in expression of one or more transcription factors. The specific roles of this increased expression are largely unknown. We show here that in mouse NIH3T3 cells, Egr-1 expression is increased two-fold 10 min after u.v. irradiation, rises to a maximum (eightfold induction) after about 2 h and then declines. The expression of p53 protein is also strongly induced but is maximal between 2 to 4 h before declining. In contrast, the expression of c-Fos, and C-Jun proteins are only slightly affected by u.v. The Egr-1 response is independent of the growth state of the cells but depends on tyrosine kinase and protein kinase C activities. c-Ha-Ras is also involved in the induction of Egr-1 in u.v. irradiated cells. Evidence presented suggests that the mechanism for the response involves oxidative stress rather than DNA damage. We show that Egr-1 functions in the protection of cells against u.v. damage since NIH3T3 cells that constitutively express antisense Egr-1 and consequently cannot produce an Egr-1 response to u.v., grow at a rate 26% less than similarly irradiated parental cells and 36% less than nonirradiated parental cells. This is the second protective role described for Egr-1.