Pott's paraplegia secondary to Scrofuloderma is a rare association.

STUDY DESIGN: Spinal tuberculosis as all other osteoarticular tuberculosis occurs as a result of hematogenous dissemination of Mycobacterium tuberculosis from a primarily infected visceral focus mainly lungs, but Pott's paraplegia occurring secondarily to Scrofuloderma has not been reported till date. PURPOSE: To document such an association of Pott's paraplegia and Scrofuloderma. METHODS: A 29-year-old female presented to us with low backache and paraplegia of 1 month duration. Clinical examination showed multiple healed Scrofuloderma lesions over sterum and neck (Figure 1). Magnetic resonance imaging (Figure 4), computed tomography (Figure 3) and X-ray (Figure 2) showed Pott's spine involving dorsal vertebrae (D8-10) with pre and paravetebral abscess with intraspinal extension. She was treated by minimally invasive surgery to drain pus and granulation tissue by resecting transverse process of D9 vertebra. Drained material was sent for histopathological examination, Zielh-neelsen (ZN) staining, culture and sensitivity for M. tuberculosis. Patient completely recovered neurologically after 1 month of surgery. RESULTS: Patient completely recovered neurologically after surgery and is presently on multidrug chemotherapy and spinal brace. CONCLUSIONS: Pott's paraplegia can occur secondary to Scrofuloderma and it can be managed by multidrug antitubercular therapy and minimally invasive surgical procedure.

Triple-negative breast cancers: an updated review on treatment options.

Morphologic features of tumour cells have long been validated for the clinical classification of breast cancers and are regularly used as a "gold standard" to ascertain prognostic outcome in patients. Identification of molecular markers such as expression of the receptors for estrogen (er) and progesterone (pgr) and the human epidermal growth factor receptor 2 (her2) has played an important role in determining targets for the development of efficacious drugs for treatment and has also offered additional predictive value for the therapeutic assessment of patients with breast cancer. More recent technical advancements in identifying several cancer-related genes have provided further opportunities to identify specific subtypes of breast cancer. Among the subtypes, tumours with triple-negative cells are identified using specific staining procedures for basal markers such as cytokeratin 5 and 6 and the absence of er, pgr, and her2 expression. Patients with triple-negative breast cancers therefore have the disadvantage of not benefiting from currently available receptor-targeted systemic therapy. Optimal conditions for the therapeutic assessment of women with triple-negative breast tumours and for the management of their disease have yet to be validated in prospective investigations. The present review discusses the differences between triple-negative breast tumours and basal-like breast tumours and also the role of mutations in the BRCA genes. Attention is also paid to treatment options available to patients with triple-negative breast tumours.

Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer.

Resistance to anti-HER2 (human epithelial growth factor receptor 2) trastuzumab therapy occurs commonly in HER2-positive breast cancer and involves overactivation of HER2 and/or AKT1. Using the model of trastuzumab-sensitive or trastuzumab-resistant HER2-positive cells with wild-type PTEN, negative regulator of AKT1, we explore the involvement of cysteine protease calpain in mechanisms of trastuzumab resistance. Overexpression of calpain1 or activation of endogenous calpain during adhesion or trastuzumab treatment of trastuzumab-sensitive cells induces cleavage of cytoplasmic domains of HER2/phospho-HER2; cleavage occurs in HER2-positive tumors. Expression of the catalytically inactive mutant of calpain1 reduces the cleavage to enhance the activity of HER2, inactivates PTEN to enhance the activation of AKT1, induces desensitization to trastuzumab and promotes survival of trastuzumab-sensitive cells. In the model of trastuzumab resistance, constitutive overactivation of HER2 and AKT1 correlates with reduced activation of calpain. Moreover, inhibitors of the catalytic site of calpain reduce the increase in constitutive activity of AKT1 and survival of trastuzumab-resistant cells selectively. Together, by regulating the activation of HER2 and PTEN/AKT1, calpain regulates trastuzumab sensitivity and survival, and the deregulation of the activation of calpain promotes trastuzumab resistance. Trastuzumab-resistant cells activate AKT1 in a mechanism dependent on the residual calpain activity, inhibition of which restores trastuzumab sensitivity and rescues resistance. These data identify calpain as a new therapeutic target in HER2-positive breast cancer.

Maspin expression inversely correlates with breast tumor progression in MMTV/TGF-alpha transgenic mouse model.

Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive activity. To date, despite the mounting evidence implicating the potential diagnostic/prognostic and therapeutic value of maspin in breast and prostate carcinoma, the lack of a suitable animal model hampers the in vivo investigation on the role of maspin at different stages of tumor progression. In this study, we used MMTV/TGF-alpha transgenic mouse model to study the expression profile of maspin in mammary tumor progression. Histopathological examinations of MMTV/TGF-alpha transgenic mice revealed TGF-alpha expression leading to hyperproliferation, hyperplasia, and occasional carcinoma in mammary gland. Interestingly, when MMTV/TGF-alpha transgenic mice were breed to homozygocity, they also developed characteristic skin papillomas. Immunohistochemistry analysis of maspin expression in the breast tissues of TGF-alpha transgenic mice showed a direct correlation between down-regulation of maspin expression and tumor progression. The loss of maspin expression was concomitant with the critical transition from carcinoma in situ to invasive carcinoma. Subsequent in-situ hybridization analyses suggest that the down-regulation of maspin expression is primarily a transcriptional event. This data is consistent with the tumor suppressive role of maspin. Furthermore, our data suggests that MMTV/TGF-alpha transgenic mouse model is advantageous for in vivo evaluation of both the expression and the biological function of maspin during the slow multi-stage carcinogenesis of mammary gland.

Temporal and quantitative regulation of mitogen-activated protein kinase (MAPK) modulates cell motility and invasion.

We have shown that ER-negative and invasive human breast cancer cell lines MDA-MB-468 and MDA-MB-231 have constitutively higher mitogen activated protein kinase (ERK1&2/MAPK) when compared to the ER-positive and non-invasive MCF-7 human breast cancer cells. In MCF-7 cells, TGFalpha stimulation induced only transient MAPK activation, leading to a transient increase in cell migration. However, MDA 231 and MDA 468 cells, TGFalpha stimulation induced sustained MAPK activation, which correlated with enhanced cell motility and in vitro invasion. Serum stimulation activates ERK/MAPK activity persistently in both ER-positive and ER-negative breast cancer cells, leading to enhanced and sustained cell migration. Inhibition of MAPK activation by anti-sense MEK expression in MDA-MB-468 cells significantly inhibits cell migration and in vitro invasion. In contrast, MCF-7 cells expressing constitutively activated MEK show a significant increase in MAPK activity and cell migration, but this failed to enhance in vitro invasion. The kinetic profiles of MAPK activation and inhibition show a relationship between the duration and magnitude of MAPK activation and cell migration in both ER-positive and ER-negative human breast cancer cells. These studies show that cell motility is modulated by the magnitude and the duration of MAPK activation; but increased activation of MAPK may not be sufficient to allow in vitro invasion in non-invasive MCF-7 breast cancer cells.

Dynamic modulation of cytoskeletal proteins linking integrins to signaling complexes in spreading cells. Role of skelemin in initial integrin-induced spreading.

Recently we showed that signaling across beta3-integrin leads to activation of calpain and formation of integrin clusters that are involved in Rac activation. The subsequent activation of Rac and Rho leads to the formation of focal complexes and focal adhesions, respectively. The goal of the present study was to determine whether different proteins link the integrin to the cytoskeleton in the different complexes. We show that talin is present in focal adhesions but not in the calpain-induced clusters. alpha-Actinin colocalized with integrin at various sites, including the calpain-induced clusters. Skelemin, a protein shown recently to interact with beta1- and beta3-integrin in vitro, colocalized with integrin in calpain-induced clusters but was absent from focal adhesions. Cells transiently expressing skelemin C2 motifs, which contain the integrin binding site, failed to form integrin clusters or to spread on a substrate for beta1- and beta3-integrins. These results 1) suggest a dynamic reorganization of integrin complexes during cell spreading, 2) show that different cytoskeletal proteins link integrins in different complexes, and 3) demonstrate that skelemin is responsible for linking integrin to the calpain-induced clusters, and 4) show that the integrin-skelemin interaction is essential for transmission of signals leading to the initial steps of cell spreading.

Preparation of pentachlorophenol derivatives and development of a microparticle-based on-site immunoassay for the detection of PCP in soil samples.

Pentachlorophenol (PCP) is used as a herbicide in agriculture and as an insecticide for termite control. Because of the apparent hazard associated with its usage, there is a need for an efficient and economic on-site screening method. A 5-min on-site test has been developed for the detection of PCP based on the OnTrak format, a successful Roche on-site test format for drugs of abuse, utilizing the principle of latex agglutination immunoassay. The test detects 1 ppm of PCP in soil samples.

Signaling across the platelet adhesion receptor glycoprotein Ib-IX induces alpha IIbbeta 3 activation both in platelets and a transfected Chinese hamster ovary cell system.

In platelets, alpha(IIb)beta(3) exists in a form that cannot bind adhesive proteins in the plasma; although it can interact with immobilized fibrinogen it cannot interact with immobilized von Willebrand factor in the vessel wall. Soluble agonists such as thrombin convert alpha(IIb)beta(3) to a form that recognizes soluble and immobilized ligands. Attempts to reconstitute alpha(IIb)beta(3) activation in a non-hematopoietic, nucleated cell system have been unsuccessful. In the present study, we have developed a transfected Chinese hamster ovary cell model in which alpha(IIb)beta(3) activation is induced by signaling across glycoprotein (GP) Ib-IX by its ligand, von Willebrand factor. GPIb-IX activates not only the transfected alpha(IIb)beta(3) but also endogenous alpha(v)beta(3). Activation of the pathways leading to integrin activation occurred even in cells transfected with GPIb-IX lacking the domain on GPIbalpha that binds 14-3-3 or that which binds actin-binding protein. These studies demonstrate that signals induced by interaction of GPIb-IX with von Willebrand factor lead to alpha(IIb)beta(3) activation and suggest that the signaling pathways by which GPIb-IX induces alpha(IIb)beta(3) activation are different to those used by thrombin. Elucidation of these differences may provide insights into therapeutic ways in which to inhibit integrin activation in selective clinical settings.

Bryostatin 1 induces ubiquitination and proteasome degradation of Bcl-2 in the human acute lymphoblastic leukemia cell line, Reh.

The ubiquitin-mediated proteolytic system has been implicated in the turnover of a number of intracellular proteins. In the present study, we investigated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquitin-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and immunoblotting analyses revealed that Bcl-2 is ubiquitinated following exposure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryostatin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the control after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 protein again rapidly decreases to 6% of its pre-bryostatin 1 level at which time a plateau is reached and maintained for another 72 h. Furthermore, ubiquitin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 treated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of Bcl-2. However, ubiquitin-Bcl-2 conjugates increase in a time-dependent manner following bryostatin 1 treatment. Lactacystin, which inhibits the proteinase activities of the proteasome, inhibited the bryostatin 1-induced decrease of Bcl-2 protein. The effect of bryostatin 1 on the proteolytic efficiency of the 26S proteasome in Reh cell extracts was also investigated and shown to increase following 1 h of bryostatin 1 treatment. Proteolytic activity reached its highest point by 3 h, and subsequently returned to control levels by 12 h, post-bryostatin 1 treatment. In addition, bryostatin 1 treatment of the Reh cell line decreased expression of bcl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. We speculate that this decrease in mRNA together with increased 26S proteolytic activity accounts for the initial rapid decrease recorded in Bcl-2 protein. These findings indicate that bryostatin 1 treatment of Reh ALL cells decreases Bcl-2 expression through two processes: a) enhanced Bcl-2 protein degradation through the activation of the ubiquitin-proteasome pathway and b) decreased bcl-2 mRNA expression.

Ruthenium bisbipyridine complexes of horse heart cytochrome c: characterization and comparative intramolecular electron-transfer rates determined by pulse radiolysis and flash photolysis.

The reaction of [Ru(bpy)2L(H2O)]2+ (bpy = 2,2'-bipyridine, L = imidazole, water) with reduced horse heart cytochrome c results in coordination of [RuII(bpy)2L] at the His 33 and His 26 sites. Coordination at the His 33 site gave a diastereomeric [RuII(bpy)2L]-His-cyt c(II) mixture favoring the lambda-Ru form regardless of the substituent on the bipyridine ligands, while substitution at the more buried His 26 site gave an isomeric distribution that varies according to the substituent on the bipyridine ligands. The diastereomeric aquoproteins (L = H2O) are distinguished by their redox potentials and their conversion to the corresponding fluorescent imidazole proteins. Intramolecular electron transfer between the reduced ruthenium bipyridine and cyt c(III) in [RuII(bpy.)(bpy)L]-His33-cyt c(III) was determined by reductive pulse radiolysis using the aqueous electron as a reducing agent, kret = (2.0 +/- 0.3) x 10(5) s-1, and kret is independent of the sixth ligand L = H2O, imidazole. In addition, the rate constant for intramolecular electron transfer from cyt c(II) to the ruthenium(III) center in [RuIII(bpy)2L]-His33-cyt c(II) was determined by oxidative pulse radiolysis using azide and carbonate radicals. This rate is very sensitive to the nature of the sixth ligand. When L = H2O, the intramolecular electron-transfer rate for the major diastereomer lambda-cis-[RuIII (bpy)2(H2O)]-His33-cyt c(II) is k = 1.1 x 10(4) s-1 and is independent of pH between 5.6 and 8.3. The minor delta-cis-[RuIII(bpy)2(H2O)]-His33-cyt c(II) isomer has pH-dependent electrochemistry and a lower rate of intramolecular electron transfer. Complete conversion from L = H2O to L = imidazole is slow, requiring more than 7 days in 1 M imidazole. A lower limit (k > 2 x 10(6) s-1) for the intramolecular electron-transfer rate constant in [RuIII(bpy)2(L)]-His33-cyt c(II), L = imidazole, could be obtained by pulse radiolysis in the absence of the slower reacting aquo species. This observation is in agreement with the value of 3 x 10(6) s-1 measured by flash photolysis. Earlier pulse radiolysis experiments primarily measured the aquoligated ruthenium protein, while the flash photolysis experiments measured the imidazole-ligated fraction because it is the only species oxidatively quenched in the photoinduced reactions. Intramolecular electron-transfer reactions for a new series of ruthenium bipyridine complexes, [Ru(dabpy)2L]-His33-cyt c proteins (dabpy = 4,4'-diamino-2,2'-bipyridine) (L = imidazole, pyridine, isonicotinamide and pyrazine), proceed with lower driving force, resulting in slower rate constants amenable to measurement by oxidative pulse radiolysis. The electron-transfer rate constants for this series spanned a wide range of the Marcus log k vs delta G plot.

The level of tyrosine kinase activity regulates the expression of p21/WAF1 in cancer cells.

It is well documented that epidermal growth factor (EGF) inhibits proliferation of A431 and MDA-468 cells via activation of p21/WAF1. In the present study, we have shown that treatment of MDA-468 and A431 cells that express high levels of EGFR with 100 ng/ml of EGF leads to 14.9-fold increase in epidermal growth factor receptor (EGFR) autophosphorylation and high levels of p21/WAF1-induction (6. 7-fold), down regulation of cdk2 activity and growth arrest. When MDA-468 or A431 cells were simultaneously treated with 100 ng/ml EGF and RG13022, a relatively specific tyrosine kinase inhibitor, there was a significant reduction in p21/WAF1 levels. In contrast, when MDA-468, A432 cells that are treated with low levels of EGF (10 ng/ml) or other cells which express low to moderate levels of EGFRs such as MCF-7, MCF-10A, MDA-231 and SKBR-3 breast cells were exposed to 100 ng/ml of EGF there was a 3.8-fold increase in EGFR autophosphorylation, leading to 1.6-fold induction of p21/WAF1 and increased cell proliferation. These results suggest that the level of EGFR tyrosine kinase activity may regulate p21/WAF1 induction in cancer cells.

Mitogen-activated protein kinase (MAPK) regulates the expression of progelatinase B (MMP-9) in breast epithelial cells.

Mitogen-activated protein kinases (MAPKs) play a major role in the mitogenic signal transduction pathway and are essential components of both growth and differentiation. Constitutive activation of the MAPK cascade is associated with the carcinogenesis and metastasis of human breast and renal cell carcinomas. The gelatinases B (MMP-9) and A (MMP-2) are 2 members of the matrix metalloproteinase (MMPs) family which are expressed in human cancers and thought to play a critical role in tumor cell invasion and metastasis. In a previous study, we have shown that EGF and amphiregulin upregulate MMP-9 in metastatic SKBR-3 cells but have no effect on MMP-2 secretion. We now investigated specific step(s) in EGF-induced signalling associated with regulation of cell proliferation and MMP-9 induction. EGF-induced signalling in SKBR-3 cells was blocked by relatively specific inhibitors either on ras (FPT inhibitor-1) or P13 kinase (Wortmannin) or by reduction in EGF-induced tyrosine kinase activity (RG 13022). Blocking these signalling pathways significantly inhibited of EGF-induced cell proliferation but only partially reduced in EGF-induced MMP-9 secretion. In contrast, when SKBR-3 cells were exposed to MEK inhibitor (PD 98059) or MAPK inhibitors (Apigenin or MAPK antisense phosphorothioate oligodeoxynucleotides), EGF-induced cell proliferation, MMP-9 induction and invasion through reconstituted basement membrane were significantly reduced. Our results suggest that interfering with MAPK activity may provide a novel means of controlling growth and invasiveness of tumors in which the signalling cascade is activated.

Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells.

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.

Dysgerminoma of the ovary: a retrospective study.

A retrospective analysis of 22 patients with ovarian dysgerminoma who were treated between 1980 and 1987 was carried out. The median age at presentation was 24.5 years. A total of 15 patients were in stage I, one patient was in stage II and six patients were in stage III. Bilateral ovarian involvement was present in four patients. Conservative surgery was carried out in nine patients and 11 patients underwent radical surgery. Two patients had biopsy only. Fourteen patients received adjuvant radiotherapy and three patients received salvage radiation for recurrent disease. The 10-year actuarial survival rate was 81.8%. All 15 patients in stage I were alive and disease-free at a median follow-up of 125 months. Four patients (one in stage II and three in stage III) died of progressive or recurrent abdominopelvic disease. Pelvic recurrence occurred after conservative surgery in two patients in stage IA who had a tumour size greater than 10 cm, but they were salvaged with radical surgery, chemotherapy and radiotherapy. There were seven patients aged 20 years or less. All were alive and disease-free at a median follow-up of 127 months.

Clinicopathologic analysis of amphiregulin and heregulin immunostaining in breast neoplasia.

We observed no association between neoplastic epithelial immunostaining for either amphiregulin (AR) or heregulin (HRG) and presence of ER, EGFR/ERBB-2 overexpression, nodal status, or disease recurrence in 34 breast carcinomas. However, stromal cell staining for both correlated with outcome; 29% of stromal cell AR - cases recurred vs. 85% for AR + cases (p = 0.001), and 41% of stromal cell HRG - cases recurred vs. 82% of HRG + cases (p = 0.01). We conclude that both HRG and AR have significant biologic roles in breast carcinoma growth or progression via mediation of host-tumor interactions which favor aggressive tumor behavior.

Efficacy and safety of granulocyte macrophage-colony stimulating factor (GM-CSF) on the frequency and severity of radiation mucositis in patients with head and neck carcinoma.

PURPOSE: Based on the clinical evidence of mucosal protection by GM-CSF during cytotoxic chemotherapy, a pilot study was undertaken to determine the safety and mucosal reaction of patients receiving GM-CSF while undergoing definitive conventional fractionated radiotherapy in head and neck carcinoma. METHODS AND MATERIALS: Patients were considered eligible if buccal mucosa and oropharynx were included in the teleradiation field. Ten adult patients with squamous cell carcinoma of head and neck (buccal mucosa--8 and posterior 1/3 tongue--2) were entered into the trial. Radiation therapy was delivered with telecobalt machine at conventional 2 Gy fraction and 5 fractions/week. The radiation portals consisted of two parallel opposing lateral fields. GM-CSF was given subcutaneously at a dose of 1 microg/kg body weight, daily, after 20 Gy until the completion of radiation therapy. Patients were evaluated daily for mucosal reaction, pain, and functional impairment. RESULTS: The median radiation dose was 66 Gy. Eight patients received > or = 60 Gy. The tolerance to GM-CSF was good. All 10 patients completed the planned daily dose of GM-CSF without interruption. Mucosal toxicity was Grade I in four patients till the completion of radiotherapy (dose range 50-66 Gy). Six patients developed Grade II reaction, fibrinous mucosal lesions of maximum size 1.0-1.5 cm, during radiotherapy. None developed Grade III mucositis. The maximum mucosal pain was Grade I during GM-CSF therapy. In two patients after starting GM-CSF the pain reduced in intensity. Functional impairment was mild to moderate. All patients were able to maintain adequate oral intake during the treatment period. Total regression of mucosal reaction occured within 8 days following completion of radiotherapy. CONCLUSIONS: GM-CSF administration concurrently with conventional fractionated radiotherapy was feasible without significant toxicity. The acute side effects of radiotherapy namely mucositis, pain, and functional impairment were nil to minimal. The results are suggestive of mucosal protection by GM-CSF during radiotherapy and warrants further study in randomized double blind trial.

Epidermal growth factor and amphiregulin up-regulate matrix metalloproteinase-9 (MMP-9) in human breast cancer cells.

The EGF family of proteins encompasses several polypeptides such as epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), amphiregulin (AR) and heregulin (HRG-beta 1). These polypeptides regulate proliferation in breast cancer cells through interaction with membrane receptors. It has been previously shown that high EGF receptor number correlates with aggressive behavior and increased metastasis in human breast cancer. In the present study, we investigated the association between EGF and EGF-like ligand-induced DNA synthesis and secretion of MMP-9 and MMP-2 in metastatic SKBR-3 and non-metastatic MCF-7 breast cancer cells. Exposure of SKBR-3 cells to EGF or AR induces expression of MMP-9 but has no effect on MMP-2 secretion. In contrast to EGF and AR, HRG had no effect on gelatinase induction. None of the EGF polypeptides had any effect on gelatinase induction in MCF-7 non-metastatic breast cancer cells. While a relatively specific inhibitor of EGF receptor tyrosine kinase, PD 153035, inhibited EGF-, AR- and HRG-induced cell proliferation, it had no effect on MMP-9 induced by EGF and AR. Experimental evidence suggests that signaling mechanisms for cell proliferation and MMP-9 induction are mediated by different pathways down-stream of EGF receptor autophosphorylation or that low levels of EGF-induced signal that escape inhibition are sufficient to induce MMP-9 but unable to support cell proliferation. In addition, our results suggest that EGF and AR may modulate invasion of metastatic breast cancer cells by increasing the expression of MMPs.

Transforming growth factor beta (TGF beta)-induced nuclear localization of apolipoprotein J/clusterin in epithelial cells.

Apolipoprotein J (apoJ)/clusterin was first identified as an 80 kDa secretory glycoprotein present in most body fluids. It has been implicated in a variety of physiological processes including cellular differentiation and apoptosis. We demonstrate here that in addition to the well characterized secreted form of the protein, there exists an intracellular, nuclear form of apoJ. This intracellular form of the protein is induced to accumulate in the nucleus of two epithelial cell lines (HepG2 and CCL64) in response to treatment with transforming growth factor beta (TGF beta). We demonstrate in vitro that apoJ protein can be translated from two in-frame ATG sites. Initiation from the first ATG encodes for the secretory form of apoJ and initiation from the second ATG, located 33 amino acids downstream of the first and lacking the hydrophobic signal sequence, encodes for a truncated apoJ protein. This shorter form of apoJ is not recognized by microsomes and therefore not glycosylated, and we postulate that it is retained intracellularly and targeted to the nucleus due to the presence of an SV40-like nuclear localization sequence (NLS). This mechanism of nuclear targeting of apoJ occurs in cells since the protein isolated from nuclei of TGF beta-treated cells and the in vitro-translated truncated form are identical by V8 protease analysis. These results suggest that the diverse physiological responses attributed to apoJ may be elicited through a common molecular mechanism involving a previously uncharacterized intracellular form of the protein.

Tyrosine kinase inhibitor as a novel signal transduction and antiproliferative agent: prostate cancer.

In prostate cancer cells, the binding of peptide growth factors to specific receptors increases tyrosine kinases (TK) activity to regulate cell proliferation, cell differentiation, and signaling processes. To determine whether inhibition of receptor TK activity inhibits tumor growth, we studied the effects of a tyrosine kinase inhibitor, RG-13022 (tyrphostin), on cultured human prostate cancer cells. RG-13022 significantly inhibited TGF alpha-induced phosphorylation of EGF receptor (EGFR). This compound inhibited TGF alpha-stimulated [3H]thymidine incorporation in a dose-dependent manner with IC50 being 30 microM. Clonogenicity in soft agar was reduced in the presence of RG-13022. Inhibitory effects were also observed in androgen-positive LNCaP cells and androgen-negative PC3 cells. RG-13022 not only inhibited TGF alpha-induced growth but also growth stimulated by epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF) and serum. In addition, RG-13022 also blocked androgen-stimulated cell proliferation, suggesting that functioning TK pathways are required for androgen-induced growth. This novel synthetic inhibitor may be useful in providing a new strategy for future therapeutic intervention for prostate cancer.