Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells.

Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological effects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our findings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.

Defective transforming growth factor beta signaling pathway in head and neck squamous cell carcinoma as evidenced by the lack of expression of activated Smad2.

PURPOSE: Transforming growth factor beta (TGF-beta) regulates cell growth and differentiation, in normal squamous epithelium, via specific TGF-beta receptors and intracellular signaling molecules (Smads). We have previously observed that TGF-beta type II receptor (TbetaR-II) expression decreases in squamous cell carcinomas as tumors become less differentiated and more biologically aggressive. However, a small fraction of tumors remain TbetaR-II positive. In this article, we examine the integrity of the other members of the TGF-beta-signaling machinery, the Smad proteins. EXPERIMENTAL DESIGN: Thirteen archived head and neck squamous cell carcinomas were selected from the files of the Pathology Department of the H. Lee Moffitt Cancer Center. Protein immunoexpression was quantitated by image analysis in the context of histopathological parameters. Mutation analysis of the MADR2/Smad2 gene was also performed. RESULTS: In both TbetaR-II-positive and TbetaR-II-negative tumors, expression of the non-TGF-beta-specific Smads (4, 6, and 7) was variable, whereas expression of the pathway-specific Smad2 was lost in 38% of the tumors. Expression of the activated, phosphorylated form of this molecule, Smad2-P, was lost in approximately 70% of the tumors. No abnormal mRNA expression and no mutations in the MADR2/Smad2 gene were observed. CONCLUSIONS: These results suggest that multiple defects in TGF-beta signaling, both at the receptor and postreceptor level, may play a role in the oncogenesis of head and neck squamous cell carcinoma.

Genetic and molecular abnormalities in tumors of the bone and soft tissues.

BACKGROUND: Malignant transformation requires the accumulation of multiple genetic alterations such as chromosomal abnormalities, oncogene activation, loss of tumor suppressor genes, or abnormalities in genes that control DNA repair and genomic instability. Sarcomas are a heterogeneous group of malignant mesenchymal tumors of difficult histologic classification and strong genetic predisposition. This article provides a comprehensive review of the cytogenetic abnormalities observed in bone and soft-tissue tumors, emphasizing known downstream molecular changes that may play a role in oncogenesis. METHODS: The database of the National Library of Medicine was searched for literature relating to genetic and molecular mechanisms in sarcomas in general and in each of the main tumor entities. RESULTS: Recent techniques in chromosome analysis and molecular cytogenetics have improved our ability to characterize genetic changes in mesenchymal tumors. Some changes are so characteristic as to be virtually pathognomonic of particular histologic types, while others are complex, difficult to characterize, and of unknown relevance to pathogenesis. The implications to the cell of some of these abnormalities are now being recognized. CONCLUSIONS: The study of sarcomas will benefit from the information derived from genetic studies and translational research. The human genome project and new methodologies, such as computer-based DNA microarray, may help in the histogenetic classification of sarcomas and in the identification of molecular targets for therapy.

Gastrointestinal stromal tumors.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. A relationship to the interstitial cells of Cajal (ICCs) has been proposed, and expression of CD117, the c-kit receptor present in ICCs, has been suggested as a marker for GISTs. METHODS: The English literature has been reviewed with an emphasis on histogenetic features, especially the potential relationship of GISTs to ICCs. RESULTS: GISTs are most common in the stomach (70%), followed by small intestine (20%), colon and rectum (5%), and esophagus (<5%). GISTs commonly have activating mutations in exon 11 (or rarely exon 9 and exon 13) of the KIT gene that encodes a tyrosine kinase receptor for the stem cell factor or mast cell growth factor. CONCLUSIONS: Malignant potential is best estimated by the simultaneous evaluation of several clinical parameters. The only absolute criterion for malignancy is tumor spread beyond the organ of origin at the time of diagnosis. The remarkable clinical response of tumors that express c-kit to treatment with the tyrosine kinase inhibitor STI571 is a triumph of molecular pharmacology.

Radiation effects on osteoblasts in vitro: a potential role in osteoradionecrosis.

OBJECTIVE: To evaluate the factors involved in bone remodeling and wound healing that may be altered by radiation therapy. DESIGN: A prospective, controlled study of biochemical activity in vitro. SUBJECTS: MC3T3-E1 mouse osteoblasts. INTERVENTIONS: Cells were irradiated at 0, 2, 4, or 6 Gy. Specimens were harvested at 1, 7, 14, 28, and 42 days following irradiation for immunohistochemical analysis of transforming growth factor beta(1) expression and transforming growth factor beta(1) type I and II receptor expression. Collagen production was measured at 1, 7, 28, 35, and 49 days after irradiation. The effects of dexamethasone on collagen production and cell proliferation were also examined. RESULTS: Irradiated cells demonstrated decreased cell proliferation and a dose-dependent, sustained reduction in collagen production when compared with control cells. An increase in transforming growth factor beta(1) type I and II receptor expression was noted in irradiated cells when compared with controls. CONCLUSION: Radiation-induced alterations of factors related to bone remodeling and wound healing have a potential role in the pathogenesis of osteoradionecrosis.

Transforming growth factor-beta type II receptors and smad proteins in follicular thyroid tumors.

OBJECTIVE: Resistance to transforming growth factor (TGF)-beta-mediated cell growth inhibition is a well-known pathogenic mechanism in epithelial neoplasia. TGF-beta signaling requires normal function of downstream mediators such as TGF-beta receptors (TbetaRs) and Smad proteins. The goal of this study is to investigate the expression of components of the TGF-beta signaling pathway in follicular tumors of the thyroid. STUDY DESIGN: Twenty follicular thyroid neoplasms were classified as adenomas (11) or minimally invasive follicular carcinomas (9) according to current pathological criteria. Protein expression was evaluated to identify differences between benign and malignant tumors that could be used as an adjunct to histopathological analysis. METHODS: Paraffin-embedded tissue sections containing tumor and adjacent nonneoplastic parenchyma were analyzed by immunohistochemistry for the expression of TbetaR type II (TbetaR-II) and Smad2, Smad4, Smad6, and Smad7. Expression of each protein in the tumor was compared with that of the corresponding adjacent nonneoplastic thyroid parenchyma. RESULTS: TbetaR-II expression was lost in 78% of the carcinomas. In the remaining 22%, TbetaR-II was preserved but Smad2 expression was lost. In all conventional adenomas, however, TbetaR-II expression was maintained. Furthermore, all tumors with normal expression of all proteins were adenomas. CONCLUSIONS: Downregulation of TbetaR-II is a consistent abnormality in follicular carcinomas and can be used to differentiate minimally invasive carcinomas from adenomas. Also, downregulation of Smad proteins is another mechanism by which carcinomas can become independent from TGF-beta-mediated growth inhibition.

Prognostic factors in malignant gastrointestinal stromal tumors.

Gastrointestinal stromal tumors are a heterogeneous group of mesenchymal neoplasms of the gastrointestinal tract in which routine histopathological evaluation fails to reveal definitive evidence of differentiation. Given the heterogeneity in clinical presentation and the frequent morphological overlap, the biological behavior of these neoplasms is difficult to predict. We have evaluated, by Cox Proportional Hazards Regression Analysis, the clinicopathological features of 51 malignant gastrointestinal stromal tumors to identify predictors of survival. In the univariate analysis, survival inversely correlated with size, number of mitoses, and patient's age. In the multivariate analysis, only the degree of necrosis and phenotypic differentiation toward smooth muscle were found to be indicators of poor prognosis. Based on these results, a simple classification scheme for gastrointestinal stromal tumors is proposed. This classification appears to have great prognostic value for these tumors, and may be useful in guiding therapeutic management.

Expression of transforming growth factor beta type II receptors in head and neck squamous cell carcinoma.

Transforming growth factor (TGF)-beta is a potent regulator of growth and differentiation in normal squamous epithelium. TGF-beta exerts its antiproliferative effect via the TGF-beta type II receptor (TbetaR-II). A decrease in TbetaR-II expression is believed to be responsible, in part, for the resistance of squamous cell carcinoma (SqCC) to the anti-proliferative effects of TGF-beta. In the present study, we used immunohistochemistry and in situ hybridization to analyze the expression of TbetaR-II along the successive oncogenic stages of head and neck squamous neoplasia, from normal epithelium to dysplasia to carcinoma. Quantitation of TbetaR-II expression in 38 SqCCs was assessed on a visual scale ranging from negative (absence of staining) to 3+ (strong staining). Normal squamous epithelium and squamous epithelium in the vicinity of the tumors showed homogenous receptor expression with moderate intensity. Dysplastic epithelium and carcinoma in situ showed a mild decrease in receptor expression intensity. Well-differentiated to moderately differentiated carcinomas showed heterogeneous expression of variable intensity, and poorly differentiated carcinomas were completely devoid of TbetaR-II. In every tumor, the superficial component showed more intense receptor expression than the invasive component. These results indicate that TbetaR-II expression inversely correlates with disease aggressiveness and suggest that aberrant TbetaR-II expression is a contributing factor to the pathogenesis of SqCC.

Predictability of PSA failure in prostate cancer by computerized cytometric assessment of tumoral cell proliferation.

OBJECTIVES: To evaluate the relationship of DNA ploidy and cell proliferation (CP) with Gleason score (GS) and clinical outcome in prostate cancer. METHODS: Sixteen patients with benign prostatic hyperplasia (BPH) and 65 patients with prostate cancer classified by GS (four groups: 2 to 4, 5 to 6, 7, and 8 to 10) were studied. All patients with carcinoma underwent prostatectomy and were separated into prostate-specific antigen (PSA) failure and nonfailure groups (failure if PSA 0.1 ng/mL or more three times after surgery). Tumoral CP (Ki-67 inmunostaining and SG2M phase) and DNA ploidy were evaluated by computerized cytometry. RESULTS: BPH were diploid with low CP (8% SG2M cells or less). Carcinomas were either diploid with high CP (greater than 8% SG2M cells) or aneuploid. CP was significantly higher (P <0.001) in tumors with GS 7 or greater than in tumors with GS less than 7 (mean percent Ki-67 cells 18.3% versus 7.8%, respectively). PSA failure increased with GS (7.1% in GS 2 to 4, 21% in GS 5 to 6, 28.6% in GS 7, and 50% in GS 8 to 10), as well as with aneuploidy (18.5% in diploid tumors versus 72.7% in aneuploid tumors). Those experiencing PSA failure had significantly higher (P <0.001) CP than those not failing (mean percent Ki-67 cells 24% and mean percent SG2M 30.4% versus 8.7% and 13.5%, respectively). Cox regression analysis showed GS, DNA ploidy, Ki-67, and SG2M to each be univariately prognostic for time to PSA failure; however, Ki-67 and SG2M were more highly significant (P <0.0001 for both) than GS (P = 0.007) or DNA ploidy (P = 0.002). After adjusting for either SG2M or Ki-67 measures of CP, neither ploidy nor GS contained additional prognostic value. CONCLUSIONS: Tumor CP and DNA ploidy can be reliably determined in prostate cancer by computerized cytometry. On the basis of our preliminary results, CP correlates well with GS and predicts PSA failure better than DNA ploidy or GS.

Cyclin D1 expression as a prognostic parameter in papillary carcinoma of the thyroid.

Papillary carcinoma of the thyroid is the most common thyroid cancer. At the time of clinical presentation, most papillary carcinomas are still confined to the thyroid gland, and appropriate surgical treatment achieves a 95% 5-year survival rate. Certain carcinomas, however, behave in a much more aggressive fashion. Because specific therapies do not exist, for those tumors that have escaped local control, patients with disseminated disease have little or no chance of permanent cure or long-term survival. Cyclin D1, a protein that plays a critical role in the control of the cell cycle, has been shown to be overexpressed in a variety of human neoplasias and may serve as a prognostic parameter of disease progression. To explore the role played by cyclin D1 in the pathogenesis of thyroid papillary carcinoma, we have quantitated, by computerized image analysis, the immunohistochemical expression of cyclin D1 in formalin-fixed, paraffin-embedded tissue from 35 conventional papillary carcinomas of the thyroid and correlated the results with established clinicopathologic parameters and available survival data.

Transforming growth factor beta receptors and p27kip in thyroid carcinoma.

OBJECTIVE: To investigate the role of cell cycle regulators in the pathogenesis of papillary carcinoma of the thyroid. DESIGN: Resistance to transforming growth factor beta-mediated inhibition is a well-known pathogenic mechanism in epithelial neoplasias. In a retrospective study, the expression of transforming growth factor beta receptors types I and II, cyclin D1, and the cyclin-dependent inhibitor p27kip, was analyzed by immunohistochemistry. Results were interpreted in the context of clinicopathological data. Patient follow-up ranged from 1 to 18 years, with a mean of 4 years. MATERIALS: Twenty conventional primary papillary carcinomas and their metastases were selected according to current pathologic criteria. Nonconventional papillary carcinomas (eg, tall-cell, columnar) were excluded from the analysis. RESULTS: Cyclin D1 was expressed more intensely in the tumor than in adjacent nonneoplastic parenchyma. Within a given tumor, however, there was significant heterogeneity in expression intensity and percentage of positive cells, particularly in metastases. Type I receptors were strongly expressed in 90% of tumors, while 80% of the tumors revealed low to no expression of type II receptors. In 10% of tumors, type I receptors were absent and type II receptors expressed. Simultaneous absence of both receptors was not observed. While p27kip was strongly expressed in nonneoplastic thyroid, it was not detected in any of the primary tumors or their metastases. CONCLUSIONS: The results strongly suggest that functional abnormalities in type II receptors result in increased levels of cyclin D1 and down-regulation of p27kip. This would maintain cells in a proliferative state and would promote tumor progression.

In situ PCR. Overview of procedures and applications.

The evaluation of gene expression in the context of cellular morphology is essential to the full understanding of cell biology. A variety of methods for detection of nucleic acids are currently available. Solution PCR requires disruption of the sample and detection of the amplified material by electrophoresis in agarose gels. In situ Hybridization methods, on the other hand, permit morphological correlation and provide a high sensitivity that is sufficient for many applications. In some instances, however, the amount of target in the sample is below the limit of detection of this technique. In situ PCR allows the detection of minimal amounts of nucleic acids with exquisite sensitivity and specificity, while the integrity of the cells and the morphology of tissues remains preserved. This technique, although not exempt from difficulties, is undergoing methodological simplifications that will make it suitable for an increasing number of basic science and clinical applications. The following is a review of the principles, methods and applications of In situ PCR.

Macrophage expression of interleukin-1 in lymphoepithelioid cell ("Lennert's") lymphoma.

Macrophage activation was studied in three cases of a genuine form of T cell non-Hodgkin's lymphoma particularly rich in epithelioid histiocytes, the so-called "lymphoepithelioid cell lymphoma" or "Lennert's lymphoma". Host tumor infiltrating macrophages actively produced Interleukin-1 as demonstrated by immunohistochemistry. The activated histiocytes also contained intracytoplasmic tumor cells which were either intact or at various stages of apoptosis. We postulate that in Lennert's lymphoma, tumor cells are capable of activating host macrophages. Initial macrophage activation is followed by IL-1 production with recruitment of additional macrophages accounting for the characteristic histological appearance of this tumor. The activated macrophages are also engaged in a phagocytic antitumoral response. Future studies should investigate if this host response can be potentiated.

Memory T cell subsets in B cell non-Hodgkin's lymphomas.

Memory T cells were quantitated by three color flow cytometry in cell suspensions from biopsy specimens of 34 B cell non-Hodgkin's lymphomas (NHL) and 10 benign lymphoid hyperplasias (BLH). CD3+CD45R0-CD45RA+ (naive), CD3+CD45R0+CDRA- (memory) and total CD3+ T cells were compared in BLH, low grade (LG), intermediate (IG) and high grade (HG) B cell NHL. Mean percentage +/- s.e. of CD45R0 + T cells for BLH, LG, IG and HG NHL were: 14.7 +/- 3.8, 11.2 +/- 3.5, 28.9 +/- 7.5 and 45 +/- 15, respectively. Mean percentage +/- s.e. of CD45RA+ T cells were: 10.2 +/- 2.6, 7.1 +/- 2.3, 5.4 +/- 1.4 and 3.5 +/- 1.2, respectively. Mean percentage +/- s.e. of total CD3+ T cells were: 42.5 +/- 11, 22.4 +/- 7.5, 39.3 +/- 10.1 and 62.7 +/- 22.2, respectively. Memory and total T cells progressively increased from LG toward IG and HG NHL while naive T cells simultaneously decreased. Although the differences in naive T cells were non-significant, the differences in memory T cells were significant between LG and IG and LG and HG NHL (both p < .0100). We previously reported an increase in activated-cytotoxic T cells in IG -HG B cell NHL. We now report that this subset of lymphocytes has also acquired memory function. Future studies should determine if in vitro expansion and adoptive transfer of this subset of T cells results in tumor cytolysis.

Analysis of apoptosis in lymph nodes of HIV-infected persons. Intensity of apoptosis correlates with the general state of activation of the lymphoid tissue and not with stage of disease or viral burden.

The occurrence of in vivo apoptosis was investigated in lymph node sections obtained from HIV-infected persons at different stages of disease. The degree of apoptosis in lymph nodes from HIV-infected individuals was compared with that observed in lymph nodes obtained from HIV-negative individuals. Apoptosis was readily detected in lymph nodes obtained from both HIV-negative and HIV-positive persons; however, the degree of apoptosis in lymph nodes obtained from HIV-positive persons was three to four times higher than that observed in the lymph nodes obtained from HIV-negative persons. In contrast to HIV-negative lymph nodes in which apoptosis was confined largely to germinal centers, in HIV-positive lymph nodes all functional compartments of the lymph node (i.e., cortex, paracortex, and sinuses) were extensively involved by this phenomenon. Furthermore, a significant correlation was observed between intensity of apoptosis and degree of activation of the lymphoid tissue associated with HIV infection. In contrast, intensity of apoptosis correlated neither with the clinical stage of HIV disease nor with the viral burden in the lymph node. Finally, apoptosis was not restricted only to CD4+ T cells; both B cells and CD8+ T cells were found to undergo apoptosis. Taken together, these results indicate that the increased intensity of the apoptotic phenomenon in HIV infection is caused by the general state of immune activation, and is independent of the progression of HIV disease and of the levels of viral load.

The steroid antagonist RU486 exerts different effects on the glucocorticoid and progesterone receptors.

To determine whether the steroid antagonist RU486 mediates its antiglucocorticoid and antiprogestin activities by the same or different receptor mechanisms, a direct comparison of RU486 interaction with glucocorticoid (GR) and progesterone (PR) receptors was made. The effects of RU486 on transformation of GR and PR 8-10S complexes in the intact cell and in vitro were analyzed by sucrose density gradient centrifugation, and the in vitro stability of receptor-heat shock protein-90 interactions was analyzed by coimmunoprecipitation. Compared to agonist, RU486 binding produced a reduction in the amount of GR converted from 8S to 4S and stabilized the GR-heat shock protein-90 complex. By contrast, PR-RU486 complexes were transformed both in vitro and in the intact cell to the same extent as receptor-agonist complexes. PR-RU486 complexes sedimented at 5-6S, whereas PR-R5020, GR-RU486, and GR-agonist complexes sedimented at 4S. The portion of GR that undergoes nuclear transformation when bound to RU486 was examined for binding to the glucocorticoid-progesterone response element of the mouse mammary tumor virus by an immunoprecipitation assay. The nuclear-transformed GR-RU486 complex bound the glucocorticoid-progesterone response element with the same affinity as the nuclear-transformed GR-triamcinolone acetonide complex. The electrophoretic mobilities of GR-RU486 complexes and GR-agonist complexes were the same, as determined by gel retardation assay. These results suggest that RU486 exerts its antiglucocorticoid activity at two levels of receptor action: prevention of complete GR transformation and alteration of a step subsequent to GR-DNA binding. As an antiprogestin, RU486 action is exerted predominantly at a post-DNA-binding step.

Gene transfer in human lymphocytes using a vector based on adeno-associated virus.

Adeno-associated virus is a nonpathogenic, dependent parvovirus that integrates at a specific site in human chromosome 19. We have used the inverted terminal repeats of the virus, which mediate integration, to establish a vector for gene transfer in human lymphocytes. A neomycin resistance gene has been stably introduced into nontransformed human T-cell clones and a subsequent analysis of the functional properties of the infected clone revealed no detectable alterations. Rescue and replication of the wild-type virus was accomplished with adenovirus superinfection; however, the vector was not rescued and did not replicate by this procedure, indicating the stability of the integrated vector and demonstrating an additional level of safety incorporated in its construction. An adeno-associated virus-based vector represents an alternative to retroviruses for gene therapy in lymphocytes.