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Purpose: This review assesses the current state of knowledge regarding preclinical and clinical pharmacology for brain tumor chemotherapy and evaluates relevant brain tumor pharmacology studies before October 2006., Results: Chemotherapeutic regimens in brain tumor therapy have often emerged from empirical clinical studies with retrospective pharmacologic explanations, rather than prospective trials of rational chemotherapeutic approaches. Brain tumors are largely composed of CNS metastases of systemic cancers. Primary brain tumors, such as glioblastoma multiforme or primary CNS lymphomas, are less common. Few of these tumors have well-defined optimal treatment. Brain tumors are protected from systemic chemotherapy by the blood-brain barrier (BBB) and by intrinsic properties of the tumors. Pharmacologic studies of delivery of conventional chemotherapeutics and novel therapeutics showing actual tumor concentrations and biologic effect are lacking., Conclusion: In this article, we review drug delivery across the BBB, as well as blood-tumor and -cerebrospinal fluid (CSF) barriers, and mechanisms to increase drug delivery to CNS and CSF tumors. Because of the difficulty in treating CNS tumors, innovative treatments and alternative delivery techniques involving brain/cord capillaries, choroid plexus, and CSF are needed.

Purpose: The incidence of primary central nervous system lymphoma (PCNSL) is increasing. Therapeutic approaches remain controversial. An animal model that mimics the clinical situation would be useful for evaluating PCNSL biology and treatment., Experimental Design: Nude rats received intracerebral (caudate nucleus, n = 49) or intraventricular (n = 4) inoculation of human B-lymphoma cell line MC116. Two to five weeks after tumor inoculation, magnetic resonance imaging (MRI) was done (n = 24), and rat brains were assessed for pathology. Five rats each received whole-brain radiotherapy (WBRT, 20 Gy) or high-dose i.v. methotrexate (3 g/m2)., Results: Intracerebral tumors developed in 84% of evaluable animals with no pretreatment, 79% of rats pretreated with 4 Gy total body irradiation, and 92% of rats pretreated with cyclophosphamide (300 mg/m2). MRI showed abnormal T2 signal and gadolinium enhancement on T1-weighted images, consistent with tumor growth 19 to 24 days after inoculation. Tumor cells staining positively for B-lymphoma markers infiltrated within the inoculated hemisphere, along fiber tracks to the contralateral hemisphere, and along the subarachnoid space and ventricles. Tumors showed reactive gliosis. Intraventricular tumor cell injection resulted in periventricular parenchymal infiltration in both hemispheres. Radiation and methotrexate were effective in vitro, but only WBRT was clearly effective after 1 week in the intracerebral model., Conclusion: This model closely mimics human PCNSL in terms of imaging, histology, and treatment sensitivity and will be useful for the development of future therapeutic strategies for PCNSL.

The optimal treatment of primary central nervous system lymphoma (PCNSL), a rare form of extranodal non-Hodgkin lymphoma, has yet to be defined. Whole-brain radiation therapy (WBRT) has limited efficacy as a single therapeutic modality and is associated with a high risk of delayed neurotoxicity. Methotrexate-based chemotherapy regimens yield poor drug penetration across the blood-brain barrier (BBB), thus necessitating administration of high doses with the concomitant risk of increased systemic and neurological toxicity. Combined-modality therapy (WBRT plus chemotherapy) can improve response and survival rates, yet it is associated with a high risk of neurotoxicity. The aim of chemotherapy in conjunction with BBB disruption is to maximize drug delivery to the brain and improve the agent's efficacy, while preserving neurocognitive function and minimizing systemic toxicity. Methotrexate-based chemotherapy regimens administered in conjunction with BBB disruption have shown promising results in PCNSL. Animal models of central nervous system lymphoma and drug neurotoxicity offer new possibilities to study the effects of various treatments on PCNSL and normal brain and can also help understand biological and pathophysiological aspects of this disease. Because the intact BBB is even less permeable to antibodies than it is to drugs, preclinical and clinical studies of monoclonal antibody delivery (for example, rituximab and 90Y ibritumomab tiuxetan) to the brain in conjunction with BBB disruption offer a new possibility to make these novel treatments more efficient against PCNSL. Regarding the evaluation of more sensitive and specific diagnostic imaging tools, iron oxide-based contrast agents for magnetic resonance imaging have shown promise for better differentiation of PCNSL from other white matter diseases.

Purpose: To assess the safety of delayed high dose intravenous (i.v.) sodium thiosulfate (STS) in a case series of 12 children with malignant brain tumors who were treated with intraarterial (i.a.) carboplatin in conjunction with blood-brain-barrier disruption (BBBD)., Methods: Twelve children ages 17 months-12 years underwent a total of 132 BBBD chemotherapy treatments and also received delayed high dose STS (i.v.). Dose 1 of STS (10-16 g/m(2)) was administered 2 or 4 hr after carboplatin, and a second STS dose was administered 4 hr after dose 1 if the child had impaired baseline hearing. Toxicity data were graded in accordance with the National Cancer Institute Common Toxicity Criteria (Version 2). Audiologic monitoring to evaluate the otoprotective potential of STS was performed on 11 children. Ototoxicity was defined in accordance with the American Speech-Language-Hearing Association (ASHA) criteria. Baseline and end of treatment hearing status were graded using Brock's criteria., Results: Nausea and vomiting were well controlled with anti-emetics administered approximately 30 min prior to STS infusion. Analogous to results in adult patients, there was mild transient hypernatremia and a trend for improved protection from ototoxicity in children who received STS delayed to 4 hr post-treatment versus 2 hr. Tumor responses were seen in heavily pre-treated patients with relatively chemo-resistant tumors, suggesting that STS did not protect the tumor from platinum cytotoxicity., Conclusion: High dose STS is well tolerated in children under 12 years of age. Further studies of STS in children are warranted to assess otoprotection and the impact of STS on platinum mediated efficacy.

Objective: Superparamagnetic iron oxide nanoparticle magnetic resonance imaging (MRI) contrast agents are gaining use in the central nervous system. The purpose of this study was to evaluate the imaging characteristics, distribution, time course, and neurotoxicity of the clinical agents ferumoxtran-10, ferumoxides, and ferumoxytol, and the laboratory preparation MION-46 in rat brain., Methods: Iron oxide agents were administered by intracerebral inoculation or intraarterially after osmotic blood-brain barrier opening in normal rats and intravenously in nude rats with intracerebral tumor xenografts. Rat brains were imaged by MRI at multiple time points and then were assessed for iron histochemistry and pathological features., Results: After intracerebral injection, MRI signal changes declined slowly over weeks to months. After transvascular delivery, transient (3 d) enhancement was seen with ferumoxtran-10 or ferumoxytol, whereas ferumoxides induced long-term (28 d) signal dropout. No pathological brain cell or myelin changes were detected after delivery of the clinical iron oxide agents to normal brains. In tumor models, ferumoxtran-10 enhanced one small-cell lung carcinoma intracerebral tumor, which correlated with iron staining in cells with macrophage morphological features at the tumor margin. Little enhancement was seen in two other models., Conclusion: These studies demonstrate the safety and efficacy of iron oxide-based MRI contrast agents in the brain and provide imaging parameters and time course data for future studies in brain tumors and neurological lesions.

Background and Purpose: Iron oxide-based contrast agents have been investigated as more specific MR imaging agents for central nervous system (CNS) inflammation. Ferumoxtran-10 is a virus-size nanoparticle, taken up by reactive cells, that allows visualization of the phagocytic components of CNS lesions. Ferumoxtran-10 was compared with standard gadolinium-enhanced MR images in this exploratory trial to assess its potential in evaluation of CNS lesions with inflammatory aspects, including lymphoma, multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), and vascular lesions., Methods: Twenty-three patients with different types of intracranial "inflammatory" lesions underwent standard brain MR with and without gadolinium, followed an average of 10 days later by a ferumoxtran-10 scan. Patients were imaged 24 hours after infusion of 2.6 mg/kg ferumoxtran-10. All MR images were evaluated subjectively by 4 investigators for a difference in enhancement patterns, which could be useful in differential diagnoses., Results: In 5 cases, (one ADEM, 2 stroke, one cavernous venous vascular malformation, one primary central nervous lymphoma) the ferumoxtran-10 scan showed higher signal intensity, larger area of enhancement, or new enhancing areas compared with gadolinium. Most MS patients showed less enhancement with ferumoxtran-10 than with gadolinium., Conclusion: Ferumoxtran-10 showed different enhancement patterns in a variety of CNS lesions with inflammatory components in comparison to gadolinium. The impact of timing and therapy need further evaluation to better assess ferumoxtran-10 in addition to gadolinium as contrast agents for use in diagnosis and monitoring therapy in patients with CNS inflammatory lesions.

Cisplatin (CDDP) is a common, highly toxic chemotherapeutic agent. This study investigates chemoprotective effects of N-acetylcysteine (NAC) and sodium thiosulfate (STS) on in vitro and in vivo CDDP toxicities. For ototoxicity studies, CDDP (6 mg/kg) was administered to rats via a retrograde carotid artery infusion. Auditory brainstem response thresholds at 4 to 20 kHz were tested before and 7 days post-treatment. STS (8 g/m(2) i.v.) was administered at 4, 8, or 12 h after CDDP. For nephrotoxicity studies, rats were treated with CDDP intraperitoneally (10 mg/kg) before or after NAC (400 mg/kg) or STS (8 g/m(2)), and blood urea nitrogen (BUN) and creatinine concentrations were measured after 3 days. In vitro cytotoxicity and chemoprotection in human tumor cell lines were assessed by cell viability and immunoblotting assays. Rats treated with STS 4 h after CDDP exhibited no hearing change. The STS 8-h group had less otoprotection, whereas 12-h rats had ototoxicity. CDDP induced high BUN and creatinine, corresponding to renal tubule toxicities. All NAC-treated animals showed normal BUN and reduced creatinine levels compared with CDDP alone and no histopathological evidence of nephrotoxicity. Delayed STS treatment was not consistently protective against nephrotoxicity. STS administration fully protected against the in vitro cytotoxic and apoptotic effects of CDDP if added within 2 h of CDDP, but chemoprotection decreased if STS administration was 4 h, and was minimal by 6 h, after CDDP. Thus, the chemoprotection route and timing of administration can be manipulated to maintain CDDP antitumor efficacy while protecting against toxicities.

Thiols such as N-acetylcysteine (NAC) are increasingly used in clinical trials of platinum chemotherapy as chemoprotectants. NAC can prevent cisdiamminedichloroplatinum (cisplatin)-induced ototoxicity, nephrotoxicity, and gastrointestinal toxicity; however, the molecular mechanisms of NAC on apoptosis and cisplatin cytotoxicity remain unknown. We investigated cisplatin cytotoxicity and NAC chemoprotection in human tumor cell lines, as assessed by immunoblotting and immunocytochemistry. Cisplatin cytotoxicity was associated with nuclear translocation of apoptosis induction factor, expression of the pro-apoptotic Bax protein, cleavage of caspases 3 and 9, and cleavage of PARP. NAC administration reversed the cytotoxic and apoptotic effects if added concurrent with cisplatin or up to 2 h after cisplatin, but chemoprotection was reduced if NAC administration was delayed more than 2 h and was minimal by 8 h after cisplatin. Expression of tumor suppressor p53 and the cell cycle regulatory protein p21 was stimulated within 5 to 10 min by cisplatin in p53-positive LX-1 small cell lung carcinoma cells, and this effect was blocked by NAC. In p53-negative SKOV3 cells, cisplatin toxicity and NAC chemoprotection remained effective, suggesting that chemoprotection may be mediated through both p53-dependent and -independent pathways. Specific kinase inhibitors demonstrated that cisplatin induced apoptosis through the p38 mitogen-activated protein kinase (MAPK) pathway, not the extracellular signal-regulated kinase MAPK pathway. These results show that NAC blocks both the death receptor and the mitochondrial apoptotic pathways induced by cisplatin. The time course for NAC chemoprotection after cisplatin matches our previous in vivo results and provides an opportunity to manipulate route and timing to maintain cisplatin antitumor efficacy while protecting against chemotherapy side effects.

Purpose: Herstatin, an autoinhibitor of the epidermal growth factor (EGF) receptor family, was evaluated for efficacy against human glioblastoma in vitro and in vivo in a rat intracranial model., Experimental Design: Glioblastoma controlled by EGF receptor (EGFR; U87MG) or by the truncated mutant, DeltaEGFR (U87MG/Delta), were transfected with Herstatin and evaluated for in vitro and in vivo growth in nude rat brain. Cells treated with purified Herstatin in vitro were evaluated for growth and signal transduction., Results: Herstatin expression prevented tumor formation by U87MG and purified Herstatin inhibited their growth in vitro in a dose-responsive fashion, whereas in vivo and in vitro growth of U87MG/Delta was resistant to Herstatin. Inhibition of U87MG growth correlated with suppressed EGF activation of EGFR and of Akt but not mitogen-activated protein kinase signaling pathways, whereas DeltaEGFR activity and intracellular signaling in U87MG/Delta were unaffected by Herstatin treatment., Conclusions: Herstatin may have utility against glioblastoma driven by the EGFR but not the mutant DeltaEGFR. Blockade of Akt but not the mitogen-activated protein kinase signaling cascade appears to be critical for suppression of intracranial tumor growth.

Objective: Ferumoxtran-10 (Combidex), a dextran-coated iron oxide nanoparticle, provides enhancement of intracranial tumours by magnetic resonance (MR) for more than 24 h and can be imaged histologically by iron staining. Our goal was to compare ferumoxtran imaging and histochemistry vs. gadolinium enhancement in malignant brain tumours on preoperative and postoperative MR., Methods: Seven patients with primary and metastatic malignant tumours underwent MR imaging with gadolinium and ferumoxtran both pre- and postoperatively. Normalized signal intensities on the ferumoxtran-enhanced scans were determined in representative regions of interest. Resected tissue from six ferumoxtran patients and from three patients who did not receive ferumoxtran was assessed for localization of iron in tumour and reactive brain., Results: All malignant tumours (all of which enhanced by gadolinium MR) showed ferumoxtran accumulation with T1 and T2 signal changes, even using a 0.15 T intraoperative MR unit in one patient. Iron staining was predominantly in reactive cells (reactive astrocytes and macrophages) and not tumour cells. In five of the seven patients, including two patients who showed additional lesions, areas enhancing with ferumoxtran but not with gadolinium were observed. Comparison of the pre- and postoperative MR revealed residual ferumoxtran-enhancing areas in four of seven cases., Conclusion: In malignant tumours, ferumoxtran may show areas of enhancement, even with a 0.15 T intraoperative MR, that do not enhance with gadolinium. Ferumoxtran-enhancing lesions have persistent increased T1 signal intensity for 2-5 days, which may provide advantages over gadolinium for postoperative imaging. Histochemistry for iron shows uptake of ferumoxtran in reactive cells (astrocytes and macrophages) rather than tumour cells.

Cisplatin (CDDP) is a widely used chemotherapeutic agent that is highly ototoxic. Animal studies and clinical trials have shown that thiosulfates can protect against platinum-induced ototoxicity. This study investigated a new model for CDDP ototoxicity in the rat, and tested the potential chemoprotective effect of administering N-acetylcysteine (NAC) before giving CDDP. Long Evans rats were treated with CDDP 6 mg/kg delivered to the aorta via a retrograde right external carotid artery infusion, 15 min after intravenous (IV) infusion of saline (n=8) or NAC 400 mg/kg (n=8), such that the vertebral arteries were perfused. Subsequent groups were similarly treated with NAC 30 min before (n=7) and 4 h after (n=7) CDDP. Auditory brainstem response (ABR) thresholds were tested at 4-20 kHz, 7 days after treatment and compared to baseline ABR values. The NAC-treated rats exhibited no significant change from baseline values at all time intervals, while the saline-treated rats showed marked ototoxicity, especially at higher frequencies. Furthermore, the rats treated with NAC 15 min before CDDP exhibited less overall toxicity to CDDP, as evidenced in weight loss 7 days post-treatment (mean for saline=-39.63 g; mean for NAC=-21.13 g; p=0.0084). These data show that treatment with NAC can prevent CDDP-induced ototoxicity in rats.

Thiol chemoprotective agents can reduce chemotherapy side effects, but clinical use is limited due to concerns of impaired chemotherapeutic efficacy. We evaluated whether an optimized bone marrow chemoprotection regimen impaired the efficacy of enhanced chemotherapy against rat brain tumors. Nude rats with intracerebral human lung carcinoma xenografts were treated with carboplatin, melphalan, and etoposide phosphate delivered intra-arterially with osmotic blood-brain barrier disruption (n = 8/group). Thiol chemoprotection was N-acetyl-L-cysteine (1000 mg/kg) 60 min before chemotherapy and/or sodium thiosulfate (8 g/m(2)) 4 and 8 h after chemotherapy, when the blood-brain barrier is reestablished. Blood counts were obtained before treatment on day 3 and at sacrifice on day 9. N-acetylcysteine serum clearance half-life was 9 to 11 min. Pretreatment with N-acetylcysteine combined with delayed administration of sodium thiosulfate protected against toxicity toward total white cells, granulocytes, and platelets (P = 0.0016). Enhanced chemotherapy reduced intracerebral tumor volume to 4.3 +/- 1.0 mm(3) compared with 29.1 +/- 4.1 mm(3) in untreated animals (P < 0.0001). Tumor volume was 3.7 +/- 0.6 mm(3) in rats that received N-acetylcysteine before and sodium thiosulfate after chemotherapy. The data indicate the efficacy of enhanced chemotherapy for rat brain tumors was not affected by thiol chemoprotection that provided excellent protection for hematological toxicity. Negative interactions of thiols with antitumor efficacy were avoided by temporal and spatial separation of chemoprotectants and chemotherapy.

Central nervous system (CNS) drainage may occur via connections to the vasculature, but in animal models up to 50% occurs via perivascular, perineural and primitive lymphatic drainage to cervical lymph nodes. We evaluated efflux of particles from the brain to cervical lymph nodes in normal rats, using Combidex iron oxide-based magnetic resonance imaging (MRI) agent. After intracerebral, intraventricular, intracarotid or intravenous injection of Combidex in normal Long Evans rats, particle localization was assessed by MRI and histochemistry for iron and the dextran coat (n = 27). Intraventricular or intracerebral injection, but not intracarotid administration of Combidex (100 micro g), resulted in MRI signal changes in the deep cervical lymph nodes around the carotid artery, and, less strongly, in the superficial cervical nodes. Within 2 h of Combidex administration, iron was histologically localized in cervical lymph nodes, with patched staining of capsule and peripheral sinus consistent with delivery via multiple afferent lymphatic vessels. Lymph node staining in groups receiving CNS Combidex was significantly different from controls (P < 0.0001) and was significantly localized in the deep vs. superficial cervical lymph nodes (P = 0.0003). The trafficking of the superparamagnetic iron particles from the CNS in the rat could be visualized by MRI and histology. Combidex provides a powerful tool to rapidly assess drainage of virus-sized particles from the CNS.

Objective: The SGN-15 monoclonal antibody-doxorubicin immunoconjugate is toxic to Lewis(Y) antigen-expressing cells and is effective against intracerebral tumors in nude rats when delivery is enhanced with osmotic disruption of the blood-brain barrier (BBB). We tested whether doxorubicin released locally in antigen-expressing cells would affect adjacent non-antigen-expressing cells in heterogeneously expressing intracerebral tumors., Methods: Nude rats with intracerebral xenografts of human small cell lung carcinoma cells with high (n = 10) or low (n = 23) Lewis(Y) antigen expression were treated with SGN-15 at a low (10 mg/kg) or high (140 mg/kg) antibody dose, administered intra-arterially with BBB disruption, and tumor volumes and antigen expression were evaluated after 6 days., Results: BBB disruption-enhanced delivery of SGN-15 (10 mg/kg) reduced the high-expressor tumor volume from 26.1 +/- 3.7 mm(3) (n = 7) in untreated control animals to 6.7 +/- 4.6 mm(3) (n = 3, P < 0.05). Untreated high-expressor tumors exhibited uniform prominent Lewis(Y) antigen staining (97.6 +/- 0.9% positive, n = 4), whereas treated tumors demonstrated areas of nil, moderate, and prominent staining (71.0 +/- 8.5% positive, n = 3, P < 0.05). In intracerebral tumors with low initial antigen expression, BBB disruption-enhanced delivery of a low dose of immunoconjugate was significantly effective, but treated tumors demonstrated low levels of antigen expression. An increase in the immunoconjugate dose did not significantly alter either efficacy or antigen expression., Conclusion: Immunoconjugate delivered across the BBB was effective against antigen-positive tumor cells, but there was not an effective chemical bystander effect against antigen-negative tumor cells.

An immunoconjugate of doxorubicin (adriamycin) and a tumor-specific monoclonal antibody, BR96-DOX (now SGN-15) targets chemotherapy to cells that express the LewisY antigen. This immunoconjugate is internalized into lysosomes in antigen-expressing cells, with release of free doxorubicin after hydrolysis of the acid-labile linker. We review our studies using BR96-DOX in a human small-cell lung carcinoma intracerebral xenograft model in nude rats. We have found that the immunoconjugate is effective against intracerebral tumors when delivery is enhanced with osmotic disruption of the blood-brain barrier (BBB). Enhanced delivery of BR96-DOX with BBB opening can work together with radiotherapy to increase antitumor efficacy, which is maximally effective if immunoconjugate is administered prior to radiotherapy. In heterogeneous brain tumors, enhanced delivery of BR96-DOX significantly reduced tumor volumes, but local release of doxorubicin by targeting antigen expressing cells shows modest cytotoxicity against adjacent non-expressor cells. Although BR96-DOX is not effective against glioma cells tested, it does provide a model for drug-immunoconjugate therapy of gliomas. Our studies in a rat brain tumor model point out the importance of optimized delivery, antigenic heterogeneity, and bystander effect for brain tumor therapy. We review additional studies of drug-mAb immunoconjugates pertinent to the treatment of gliomas.

The November 2000 NIH report of the Brain Tumor Progress Review Group identified delivering and targeting therapeutic agents as a priority in the treatment of malignant brain tumors. For this reason, the seventh annual Blood-Brain Barrier Disruption Consortium meeting, partially funded by an NIH R13 Grant, focused on recent advances in targeted delivery to the central nervous system, clinical trials for primary and metastatic brain tumors using enhanced chemotherapy delivery, and strategies to lessen the toxicities associated with dose intensive treatments, using thiols.

Background and Purpose: Ultrasmall superparamagnetic iron oxide particles result in shortening of T1 and T2 relaxation time constants and can be used as MR contrast agents. We tested four hypotheses by evaluating MR images of intracranial tumors after infusion of two iron oxide agents in comparison with a gadolinium chelate: 1) Ferumoxtran in contrast to ferumoxides can be used as an intravenous MR contrast agent in intracranial tumors; 2) ferumoxtran enhancement, albeit delayed, is similar to gadolinium enhancement; 3) ferumoxtran-enhanced MR images in contrast to gadolinium-enhanced MR images may be compared with histologic specimens showing the cellular location of iron oxide particles; 4) ferumoxtran can serve as a model for viral vector delivery., Methods: In 20 patients, ferumoxides and ferumoxtran were intravenously administered at recommended clinical doses. MR imaging was performed 30 minutes and 4 hours after ferumoxides infusion (n = 3), whereas ferumoxtran-enhanced MR imaging (n = 17) was performed 6 and 24 hours after infusion in the first five patients and 24 hours after infusion in the remaining 12. MR sequences were spin-echo (SE) T1-weighted, fast SE T2- and proton density-weighted, gradient-recalled-echo T2*-weighted, and, in four cases, echo-planar T2-weighted sequences. Representative regions of interest were chosen on pre- and postcontrast images to compare each sequence and signal intensity., Results: Despite some degree of gadolinium enhancement in all tumors, no significant T1 or T2 signal intensity changes were seen after ferumoxides administration at either examination time. Fifteen of 17 patients given ferumoxtrans had T1 and/or T2 shortening consistent with iron penetration into tumor. Histologic examination revealed minimal iron staining of the tumor with strong staining at the periphery of the tumors., Conclusion: 1) Ferumoxtran can be used as an intravenous MR contrast agent in intracranial tumors, mostly malignant tumors. 2) Enhancement with ferumoxtran is comparable to but more variable than that with the gadolinium chelate. 3) Histologic examination showed a distribution of ferumoxtran particles similar to that on MR images, but at histology the cellular uptake was primarily by parenchymal cells at the tumor margin. 4) Ferumoxtran may be used as a model for viral vector delivery in malignant brain tumors.

Modulation of thiol levels may alter both the efficacy and toxicity of chemotherapeutic agents. We investigated cytoenhancement, using L-buthionine-[S,R]-sulfoximine (BSO) to reduce cellular glutathione levels prior to intracarotid alkylator administration. We also evaluated chemoprotection against chemotherapy-induced systemic toxicity when the thiol agents N-acetylcysteine (NAC) and sodium thiosulfate were administered into the descending aorta to limit brain delivery. BSO treatment reduced rat brain and intracerebral tumor glutathione levels by 50-65%, equivalent to the reduction in liver and s.c. tumor. BSO treatment significantly enhanced the toxicity of chemotherapy with carboplatin, melphalan, and etoposide phosphate against granulocytes, total white cells, and platelets. Intracarotid administration of NAC resulted in high delivery to the brain, whereas infusion via the descending aorta minimized brain delivery. When NAC, with or without sodium thiosulfate, was administered via aortic infusion prior to chemotherapy, the magnitude of the bone marrow toxicity nadir was minimized, even with BSO-enhanced myelosuppression. Thus, BSO depleted brain and brain tumor glutathione but thereby increased chemotherapy-induced myelosuppression. Surprisingly, although NAC was found to readily cross the blood-brain barrier when given into the carotid artery, aortic infusion of NAC resulted in minimal exposure to the central nervous system (CNS) vasculature because of rapid clearance. As a result, aortic infusion of NAC to perfuse bone marrow and minimize myelosuppression and toxicity to visceral organs could be performed without interfering with the CNS cytotoxicity of intracarotid alkylators, even after BSO depletion of CNS glutathione.

Modulation of glutathione has been proposed as a mechanism to alter the efficacy and toxicity of chemotherapeutic agents. We investigated in vitro cytoenhancement of chemotherapy toxicity by reducing cellular glutathione levels with L-buthionine-[S,R]-sulfoximine (BSO), and chemoprotection with small molecular weight sulfur-containing agents that mimic or replace glutathione. Cytotoxicity, caspase-2 enzymatic activity, and in situ DNA staining for apoptosis were assessed in cultured human small cell lung carcinoma cells and fibroblasts. BSO treatment reduced the half-maximal cytotoxic dose of the alkylating chemotherapeutics melphalan, carboplatin, and cisplatin, and increased the total magnitude of cell death. Melphalan was more sensitive than carboplatin or cisplatin to BSO. The chemoprotective agents sodium thiosulfate, N-acetylcysteine, and glutathione ethyl ester reduced the cytotoxicity of all three alkylating chemotherapeutics regardless of BSO treatment, but D-methionine was effective only against the platinum agents. N-Acetylcysteine was the most effective protectant tested. Chemoprotection against melphalan toxicity was maximally effective only if administered concurrent with chemotherapy, whereas chemoprotection for the platinum agents remained effective if delayed 4 h after chemotherapy. BSO enhancement and N-acetylcysteine chemoprotection for melphalan toxicity occurred at least partially through an apoptotic mechanism. Modulation of glutathione levels will be valuable in the clinical setting if chemotherapy and chemoprotectant can be physically and/or temporally separated. Cytoenhancement and chemoprotection may be particularly useful in the central nervous system where the blood-brain barrier of the cerebral vasculature creates two compartments, for cytoenhancement in brain tumors and systemic chemoprotection.

Carboplatin is effective in the treatment of malignant brain tumors. However, when administered in conjunction with osmotic opening of the blood-brain barrier (BBB), carboplatin is ototoxic. The purpose of this study was to determine whether delayed administration of sodium thiosulfate (STS), given after BBB closure, provided protection against carboplatin ototoxicity. Patients underwent monthly treatment with intra-arterial carboplatin (200 mg/m2/day x 2) in conjunction with osmotic opening of the BBB, for up to 1 year. Audiological assessment was conducted at baseline and within 24 h before each monthly treatment. STS was administered i.v. as one (20 g/m2) or two (20 g/m2 and 16 g/m2) 15-min doses, depending on baseline hearing status. The initial group received the first STS dose 2 h (or 2 and 6 h) after carboplatin (STS2) and a subsequent group received STS 4 h (or 4 and 8 h) after carboplatin (STS4). Audiological data were compared with a historical comparison group (HCG) treated with carboplatin without STS. Spearman correlation coefficients comparing STS 2 (n = 24), STS4 (n = 17), and HCG (n = 19) indicated significantly lower rates of ototoxicity with increased delay in STS (P = 0.0006). On the basis of the analysis of hearing levels, there were significant differences among the two STS groups and HCG at 8000 Hz (P = 0.0010) and at 4000 Hz (P = 0.0075). The log-rank test for time to ototoxicity indicated a significant difference between STS4 and HCG (P = 0.0018). Delayed STS was effective in protecting against carboplatin-induced hearing loss. STS delayed to 4 h after carboplatin significantly decreased time to development of ototoxicity and rate of ototoxicity when compared with HCG.

Background: The aim of this study was to determine the safety and efficacy of intraarterial chemotherapy with osmotic opening of the blood-brain barrier (BBB) for the treatment of malignant brain tumors when administered across multiple centers., Methods: Patients with primary central nervous system lymphoma (PCNSL), primitive neuroectodermal tumor (PNET), germ cell tumor, cancer metastasis to the brain, or low or high grade glioma were eligible. Prior to entry, magnetic resonance imaging or computed tomography brain scan, medical history, neurologic status, and Karnofsky performance status were reviewed at the coordinating center. Standardized anesthesia and intraarterial catheterization guidelines were followed by a multidisciplinary team at each center. Between March 1994 and November 1997, 5 universities treated 221 adult patients with intraarterial chemotherapy with or without osmotic opening of the BBB (2464 procedures)., Results: Of evaluable patients with PCNSL, 40 of 53 (75%) achieved complete response (CR). All evaluable patients with PNET (n = 17), metastatic disease (n = 12), or germ cell tumor (n = 4) achieved stable disease (SD) or better. Of 57 evaluable patients with glioblastoma multiforme, 45 (79%) achieved SD or better. Asymptomatic subintimal tear occurred in 11 of 221 patients (5%), pulmonary embolism in 6 of 221 (2.7%), and renal toxicity in 4 of 221 (1.8%). One patient with extensive glioma expired within 48 hours after treatment., Conclusions: Using standard guidelines and protocols, intraarterial chemotherapy with or without osmotic opening of the BBB is feasible across multiple centers with a low incidence of catheter-related complications. In patients with chemotherapy-sensitive tumors, such as PCNSL, PNET, germ cell tumor, and cancer metastasis to the central nervous system, enhanced delivery results in a high degree of tumor response, with an efficacy profile that is reproducible across multiple centers., (Copyright 2000 American Cancer Society.)

Platinum-based chemotherapeutic agents, such as carboplatin and cisplatin, are effective against many human tumors, but their use may be limited by a high incidence of ototoxicity. Delayed administration of the chemoprotective agent sodium thiosulfate (STS) reduces the ototoxicity of carboplatin in a guinea-pig model, when given up to 8 h after the chemotherapy, and also reduces hearing loss in patients given carboplatin with osmotic blood-brain barrier opening for treatment of brain tumors. We tested whether STS, given at times that achieved otoprotection, could impact the chemotherapeutic efficacy of carboplatin. The impact of STS was evaluated by measuring the onset of growth of LX-1 human small cell lung carcinoma s.c. xenografts in the nude rat. When STS was administered as two boluses, 2 and 6 h after treatment with carboplatin and etoposide, there was a decrease in the time to tumor progression. In contrast, when STS administration was delayed until 8 h after carboplatin/etoposide, there was no reduction in the antitumor cytotoxicity of the chemotherapy. STS infusion did not significantly affect ultrafilterable platinum pharmacokinetics in the guinea pig. To explore the potential wider applicability of STS, in a pilot study we tested its efficacy against cisplatin ototoxicity. Delayed administration of STS, 2 h after cisplatin, was protective against cisplatin-induced ototoxicity in the guinea pig model, as determined by electrophysiological measures. On the basis of these data, we suggest that delayed administration of STS may provide a mechanism to reduce the ototoxicity caused by administration of carboplatin or cisplatin for both central nervous system and systemic cancer chemotherapy.

Background and Purpose: Osmotic disruption of the blood-brain barrier (BBB) provides a method for transvascular delivery of therapeutic agents to the brain. The apparent global delivery of viral-sized iron oxide particles to the rat brain after BBB opening as seen on MR images was compared with the cellular and subcellular location and distribution of the particles., Methods: Two dextran-coated superparamagnetic monocrystalline iron oxide nanoparticle contrast agents, MION and Feridex, were administered intraarterially in rats at 10 mg Fe/kg immediately after osmotic opening of the BBB with hyperosmolar mannitol. After 2 to 24 hours, iron distribution in the brain was evaluated first with MR imaging then by histochemical analysis and electron microscopy to assess perivascular and intracellular distribution., Results: After BBB opening, MR images showed enhancement throughout the disrupted hemisphere for both Feridex and MION. Feridex histochemical staining was found in capillaries of the disrupted hemisphere. Electron microscopy showed that the Feridex particles passed the capillary endothelial cells but did not cross beyond the basement membrane. In contrast, after MION delivery, iron histochemistry was detected within cell bodies in the disrupted hemisphere, and the electron-dense MION core was detected intracellularly and extracellularly in the neuropil., Conclusion: MR images showing homogeneous delivery to the brain at the macroscopic level did not indicate delivery at the microscopic level. These data support the presence of a physiological barrier at the basal lamina, analogous to the podocyte in the kidney, distal to the anatomic (tight junction) BBB, which may limit the distribution of some proteins and viral particles after transvascular delivery to the brain.

Objective: To compare transient blood-brain barrier disruption (BBBD) by hypertonic mannitol with pharmacological modification of the blood-tumor barrier by the vasoactive peptide bradykinin for delivery of small and large agents to nude rat intracerebral xenografts., Methods: Female nude rats (n = 104) with 6-day intracerebral human small cell lung carcinoma tumors were treated using BBBD (n = 24), intracarotid bradykinin (n = 38), or saline (controls, n = 32) administered intra-arterially. During or immediately after infusion, the rats were given radiolabeled agent (methotrexate or dextran 70; Dupont NEN, Boston, MA). The rats were killed 10 minutes later, and samples of tumor and brain regions were obtained for scintillation counting. Twenty-two additional rats were examined using magnetic resonance imaging after administering one of two contrast agents (gadoteridol or iron oxide nanoparticles) or saline (controls) in conjunction with BBBD or bradykinin., Results: After BBBD, the delivery of both small (methotrexate) and large (dextran 70) radiolabeled tracers was increased 2- to 6-fold in the tumor and 3- to 20-fold in surrounding brain, as compared with saline controls. After bradykinin treatment, there was minimal change in delivery of methotrexate or dextran 70 to tumor and brain around tumor, with the greatest increase less than 60% over controls. Magnetic resonance imaging demonstrated increased delivery of both small and large contrast agents to the treated hemisphere after BBBD. In comparison, no increased tumor enhancement could be detected after bradykinin treatment., Conclusion: BBBD resulted in global delivery of a variety of agents in a wide range of sizes. In this human brain tumor xenograft model, bradykinin was not effective at increasing delivery to the tumor of any agent tested.

Sodium thiosulfate (STS) provides protection against carboplatin-induced ototoxicity in an animal model. The purpose of this study was to determine the STS dose required for otoprotection, in patients with malignant brain tumors treated with carboplatin in conjunction with osmotic blood-brain barrier disruption. Twenty-nine patients received STS intravenously 2 hr after carboplatin. Doses were escalated from 4 g/m2 to 8, 12, 16 and 20 g/m2 on consecutive months. Audiologic assessment was performed at baseline and monthly. The audiograms were compared with those of 19 similarly treated historical control patients who did not receive STS. The incidence of ototoxicity in the historical control group of patients was 79% (15/19). This group had an average loss of 20.8 +/- 5.9 dB (n = 19) at 8 kHz after one treatment with carboplatin, whereas the STS treatment group lost only 3.7 +/- 2 dB (n = 15) after one treatment. This difference was statistically significant as assessed by Student's t test (P < .05). Furthermore, patients in the STS treatment group with excellent base-line hearing showed little change in hearing thresholds at 8 kHz after the second treatment (8.0 +/- 8.3 dB) (n = 5) compared with the historical control patients with excellent base-line hearing, (40.5 +/- 8.6 dB) (n = 11). Our data support that doses of 16 or 20 g/m2 of STS decrease carboplatin-induced hearing loss without central nervous system entry. Clinical demonstration of an otoprotective effect with a two-compartment system to prevent drug-induced hearing loss, while preserving central nervous system cytotoxicity, has not been reported previously.

The volume of distribution in tissue (Vt) that can be achieved by direct interstitial infusion of therapeutic agents into brain is limited. The maintenance of a pressure gradient during interstitial infusion to establish fluid convection has been shown to increase the Vt of small, medium, and large molecules. We have used monocrystalline iron oxide nanocompounds, superparamagnetic particles of sizes the same order of magnitude as virions, to investigate the effect of dose, the volume of infusate, and the time of infusion on the distribution of large molecules in rodent brain. Our initial study in rats (n = 6) replicated the results of a previously described report of convection-enhanced delivery in cats. At a constant rate and concentration, the Vt increased in a linear fashion, proportional to the increases in time, volume, and dose. When using a constant rate and a constant concentration, however, it is unclear which variable or variables (dose, volume, infusion time) have the greatest influence on this effect. Therefore, we assessed each variable independently (n = 12). When the iron dose was increased from 5.3 to 26.5 micrograms, there was a three- to fivefold increase in the Vt, depending on the volume and time of infusion (2 Microliters/20 min, 24 microliters/20 min, or 24 microliters/120 min) (P < 0.001). When the volume of infusate was increased from 2 to 24 microliters, at an infusion time of 20 minutes and a dose of either 5.3 or 26.5 micrograms, there was a 43 or 52% decline in the Vt, respectively (P = 0.018). When the time for the infusion of 24 microliters was increased from 20 to 120 minutes, there was a 79% increase in the Vt at a dose of 26.5 micrograms but no change in the Vt at a dose of 5.3 micrograms. The effect associated with infusion time was not significant (P = 0.113). Magnetic resonance imaging was performed to document the distribution of monocrystalline iron oxide nanocompounds in vivo, and histochemical staining for iron was used to document the distribution of monocrystalline iron oxide nanocompounds in tissue sections. The Vt for both methods was calculated by computer image analysis, and the correlation between magnetic resonance and histological volumes was determined (r2 = 0.93). On the basis of this model, we suggest that dose, rather than convection, might be the most important variable in maximizing the Vt and improved distribution might be achieved by administering an increased concentration of agent.

When carboplatin (cis-diammine-1,1-cyclobutane-dicarboxylato-platinum) delivery to brain tumors is optimized with osmotic blood-brain barrier disruption (BBBD), high frequency hearing loss can result. Treatment with sodium thiosulfate (STS) blocked carboplatin cytotoxicity against the LX-1 human small cell lung carcinoma cell line in vitro. STS decreased carboplatin-induced ototoxicity in a guinea pig model, as determined by electrophysiological measurements and analysis of inner ear outer hair cell numbers. Protection was found when STS was administered up to 8 h subsequent to carboplatin but not 24 h after carboplatin. In a rat model of osmotic BBBD, STS was neurotoxic when given immediately after BBBD but not when given 60 min after BBBD, when the barrier is reestablished. Thus, delayed administration of STS may provide a mechanism to reduce the cochlear toxicity caused by BBBD-enhanced carboplatin delivery to the brain.

Delivery of adenovirus, herpes simplex virus (HSV), and paramagnetic monocrystalline iron oxide nanoparticles (MION) to rat brain (n = 64) was assessed after intracerebral inoculation or osmotic disruption of the blood-brain barrier (BBB). After intracerebral inoculation, the area of distribution was 7.93 +/- 0.43 mm2 (n = 9) for MION and 9.17 +/- 1.27 mm2 (n = 9) for replication-defective adenovirus. The replication-compromised HSV RH105 spread to 14.00 +/- 0.87 mm2 (n = 8), but also had a large necrotic center (3.54 +/- 0.47 mm2). No infection was detected when virus was administered intra-arterially without hyperosmotic mannitol. After osmotic BBB disruption, delivery of the viruses and MIONs was detected throughout the disrupted cerebral cortex. Positive staining was found in 4 to 845 cells/100 microns thick coronal brain section (n = 7) after adenovirus administration, and in 13 to 197 cells/section (n = 8) after HSV administration. Cells of glial morphology were more frequently stained after administration of adenovirus, whereas neuronal cells were preferentially stained after delivery of both HSV vectors and MION. In a preliminary test of vector delivery in the feline, MION was detected throughout the white matter tracts after inoculation into normal cat brain. Thus MION may be a tool for use in vivo, to monitor the delivery of virus to the central nervous system. Additionally, BBB disruption may be an effective method to globally deliver recombinant viruses to the CNS.

The delivery of viral vectors to the brain for treatment of intracerebral tumors is most commonly accomplished by stereotaxic inoculation directly into the tumor. However, the small volume of distribution by inoculation may limit the efficacy of viral therapy of large or disseminated tumors. We have investigated mechanisms to increase vector delivery to intracerebral xenografts of human LX-1 small-cell lung carcinoma tumors in the nude rat. The distribution of Escherichia coli lacZ transgene expression from primary viral infection was assessed after delivery of recombinant virus by intratumor inoculation or intracarotid infusion with or without osmotic disruption of the blood-brain barrier (BBB). These studies used replication-compromised herpes simplex virus type 1 (HSV; vector RH105) and replication-defective adenovirus (AdRSVlacZ), which represent two of the most commonly proposed viral vectors for tumor therapy. Transvascular delivery of both viruses to intracerebral tumor was demonstrated when administered intraarterially (i.a.) after osmotic BBB disruption (n = 9 for adenovirus; n = 7 for HSV), while no virus infection was apparent after i.a. administration without BBB modification (n = 8 for adenovirus; n = 4 for HSV). The thymidine kinase-negative HSV vector infected clumps of tumor cells as a result of its ability to replicate selectively in dividing cells. Osmotic BBB disruption in combination with i.a. administration of viral vectors may offer a method of global delivery to treat disseminated brain tumors.

The Korat cat provides an animal model for type II GM2-gangliosidosis (Sandhoff disease) that may be suitable for tests of gene replacement therapy with the HEXB gene encoding the beta subunit of the beta-hexosaminidases. In the present report, we examined the brain and liver pathology of a typical Sandhoff-affected cat. We characterized the feline HEXB complementary DNA (cDNA) and determined the molecular defect in this feline model. cDNA libraries were produced from one normal and one affected animal, and cDNA clones homologous to human HEXB were sequenced. In the affected cDNA clone, the deletion of a cytosine residue at position +39 of the putative coding region results in a frame shift and a stop codon at base +191. This disease-related deletion was consistently detected by sequencing of cloned polymerase chain reaction amplified reverse transcribed messenger RNA from one more normal Korat and two additional affected animals. The defect was further demonstrated using single-strand conformational polymorphism analysis of the polymerase chain reaction products. In addition, alternative splicing of both normal and affected messenger RNAs was demonstrated. These results should facilitate the use of this animal model to assess gene therapy.

The 21-amino acid mammalian peptide endothelin (ET) is a powerful vasoconstrictor, a mitogen for fibroblasts and vascular smooth muscle cells, and a potent effector for numerous tissues. Through extracellular interaction with G protein-coupled transmembrane receptors, ET stimulates intracellular second messenger events that in turn activate immediate early gene transcription. Using Northern blot hybridization and nuclear run-on analyses, we examined the modulation of c-fos, fos-B, fra-1, c-jun, and jun-B gene transcripts in Rat-1 fibroblasts after ET treatment. Furthermore, we investigated the role that intracellular Ca2+ transients played in effecting this gene regulation, using the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to block Ca(2+)-dependent transcription. Our results demonstrate that ET rapidly effects increased RNA levels for all five fos/jun family genes investigated, at least two of them by increasing gene transcription. Furthermore, our results argue that increased intracellular free Ca2+ is directly involved in the induction of these fos/jun family genes by ET. While mobilization of intracellular Ca2+ is not the only pathway to fos/jun gene induction used by ET, it is clearly a major component of the signaling apparatus that is set in motion by this potent effector.

Changes in intracellular Ca++ levels are observed as a second messenger in response to a number of cellular agonists, including epidermal growth factor, transforming growth factor beta 1, and endothelin-1. The role of elevated intracellular Ca++ in transducing the effects of these three agonists on gene expression has been studied using two target genes: transin/stromelysin-1 and the endogenous murine retrovirus VL30. Although the effects of EGF and TGF beta 1 on transin/stromelysin-1 mRNA expression appear to be independent of these agonists' effects on intracellular Ca++ levels, elevated Ca++ interacted synergistically with activators of pkC to induce transin expression, even though neither agent alone could induce transin/stromelysin-1 expression. In contrast, the integrated VL30 retrovirus could be induced by Ca++ ionophores alone, and induction of VL30 mRNA by other agonists was blocked if intracellular Ca++ levels were held below a threshold value of 165 nM with Ca++ chelators. Genetic analysis of the VL30 upstream regulatory region indicated that a triple-repeat element present in the VL30 long-terminal repeat could function as an inducible enhancer, but responsiveness to either EGF or pkC activation required the concomitant elevation of intracellular Ca++. Because EGF was capable of inducing expression even in pkC-depleted cells, providing Ca++ levels were elevated, these results indicate that elevated intracellular Ca++ is capable of interacting synergistically with multiple signaling pathways to stimulate increased gene expression.

Endothelin-1 (ET-1) has been shown to require Ca2+ influx for activation of vascular smooth muscle in vivo, but in vitro models show that ET-1 mobilizes intracellular Ca2+ and is independent of extracellular Ca2+. We present data that suggest ET-1 modulates cellular responses through a dual mechanism involving both phosphatidylinositol turnover and Ca2+ channel activation. Addition of low concentrations of ET-1 (less than 10(-9) M) to serum-deprived quiescent Rat-1 cells stimulated Ca2+ influx while having little effect on diacylglycerol (DG) release or intracellular Ca2+ levels. In contrast, higher concentrations of ET-1 (greater than 10(-9) M) stimulated intracellular Ca2+ transients and release of inositol trisphosphate (IP3) and DG but did not activate Ca2+ uptake. Stimulation of Ca2+ influx at low [ET-1] could not be accounted for by depletion of intracellular IP3-sensitive pools. Neither the stimulation of Ca2+ influx at low [ET-1] nor the inhibitory actions of high [ET-1] could be mimicked by the activation of protein kinase C. We tested the hypothesis that elevated intracellular Ca2+ was inhibitory for Ca2+ influx. When intracellular Ca2+ transients were maintained below approximately 165 nM by chelation with BAPTA or BAPTA derivatives with altered affinity for Ca2+, Ca2+ influx was stimulated over the entire range of ET-1 concentrations. In addition, experimentally elevating intracellular Ca2+ levels with the tumor promoter thapsigargin abolished ET-1-stimulated Ca2+ influx. These data suggest that the biological consequences of ET-1 release may be determined by local concentration differences. Thus in vascular smooth muscle cells ET-1 may act either to mobilize intracellular Ca2+ or to promote Ca2+ influx, depending on the distance from the endothelial cell source in the vascular wall. The activation of different processes by low and high ET-1 concentrations may determine the physiological response to ET-1 stimulation in vivo.

In addition to its powerful vasoconstrictive activity, endothelin-1 (ET-1) is a potent agonist for stimulating a multitude of second messenger pathways. In the Rat-1 fibroblastic cell line, ET-1 induces a robust elevation of the intracellular levels of Ca2+, diacylglycerols (DGs), and inositol trisphosphate (IP3). Although low concentrations of ET-1 stimulate a significant increase in the rate of Ca2+ influx, this Ca2+ influx is not required for the observed increases in either IP3 or DG levels following ET-1 treatment, as both of these effects are observed even in the absence of extracellular Ca2+. The ability of ET-1 to stimulate Ca2+ influx shows a biphasic pattern, such that Ca2+ influx is stimulated at low ET-1 concentrations and inhibited at high concentrations. Investigations of the molecular mechanisms underlying this biphasic response indicate that elevated intracellular Ca2+ levels exert a negative feedback inhibition on Ca2+ influx, which can be relieved by the chelation of intracellular Ca2+. The ability of ET-1 to activate a number of distinct signal transduction pathways appears to have direct functional significance in determining the targeting of ET-1 activation. Short-term effects of ET-1 stimulation such as the induction of gene expression appear to be independent of ET-1's ability to activate protein kinase C (PKC) by elevating DG levels, as depletion of PKC activity has little or no effect on gene expression. In contrast, the ability of ET-1 to induce the rapid expression of the VL30 gene is totally dependent upon the ability of ET-1 to elevate intracellular Ca2+ levels above a specific threshold. Activation of PKC by ET-1, however, is essential for the long-term effects of ET-1 on cell proliferation and anchorage-independent growth, as the ability of ET-1 to promote DNA synthesis and to synergize with epidermal growth factor in augmenting anchorage-independent growth is significantly inhibited by prior PKC depletion. Thus, in fibroblasts, ET-1 appears to activate at least two bifurcating pathways: a Ca(2+)-sensitive pathway involved in the regulation of gene expression, and a PKC-dependent pathway required for the mitogenic effects of ET-1.

The transcriptionally active RVL3-VL30 element contains a triple repeat of TGACTCC, a sequence nearly identical to the AP-1 binding site. However, 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation was unable to elicit chloramphenicol acetyltransferase (CAT) expression from a construct containing these AP-1-like sequences upstream of the thymidine kinase promoter present in pTES. Endothelin, which activates protein kinase C (pkC) and elevates intracellular Ca2+ in Rat-1 cells, was effective in stimulating CAT expression from the VL30-pTES construct. We attempted to assess the relative importance of these second messenger systems by stimulating each pathway separately with exogenous agonists. We determined that neither stimulation of pkC by the tumor promoter TPA nor elevation of intracellular Ca2+ by the tumor promoter thapsigargin was sufficient to stimulate CAT expression from the VL30-pTES vector. When combined, the two tumor promoters induced a synergistic increase in CAT expression. Our data indicate that elevation of intracellular Ca2+ by thapsigargin was not required for full activation of pkC by TPA. First, TPA was able to stimulate expression of other genes in Rat-1 cells, indicating full activation of pkC. Second, thapsigargin synergized effectively with epidermal growth factor to stimulate CAT activity from the VL30-pTES construct in cells depleted of pkC activity by chronic TPA treatment. The permissive effects of thapsigargin on gene expression were also observed for an endogenous gene, transin/stromelysin. The permissive effects of elevated intracellular Ca2+ levels may represent a general mechanism for the stimulation of some genes by pkC-mediated pathways.

We investigated the role of intracellular [Ca2+] in mediating the independent signal transduction pathways leading to induction of VL30 RNA expression by multiple agonists in the Rat-1-derived RVL-3 cell line. This cell line contains a single integrated VL30 element, and displays a rapid transcriptional activation of VL30 following stimulation by epidermal growth factor, endothelin, or the phorbol ester tumor promoter 12-O-tetradecanoylphorbol acetate (Rodland, K. D., Brown, A. M. C., and Magun, B. E. (1987) M. Cell. Biol. 7, 2296-2298). Neither epidermal growth factor nor endothelin is dependent upon protein kinase C for activation of VL30 expression, as both of these agonists induce normal levels of VL30 RNA expression, even in cells which have been severely depleted of protein kinase C following chronic 12-O-tetradecanoylphorbol acetate exposure. Induction of VL30 RNA expression by either endothelin or 12-O-tetradecanoylphorbol acetate was blocked by concomitant exposure of RVL-3 cells to the intracellular Ca2(+)-chelating agent 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid at a concentration sufficient to buffer intracellular [Ca2+] below 200 nM, and VL30 RNA was induced by the application of the Ca2+ ionophore A23187 in the absence of agonist. Normal levels of VL30 expression in response to epidermal growth factor were observed at 165 nM [Ca2+], but were significantly inhibited at 115 nM [Ca2+]. Both the protein kinase C-dependent and protein kinase C-independent pathways leading to VL30 transcription were dependent upon the presence of an intracellular [Ca2+] exceeding 115 nM. The dependence upon intracellular Ca2+ transients for transcriptional induction by endothelin appears to be a characteristic of VL30 expression, as 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid treatment did not prevent the endothelin-induced transcription of the protooncogenes c-jun and c-fos.

Transforming growth factor beta (TGF-beta), initially identified in platelet extracts by virtue of its ability to confer anchorage-independent growth and a neoplastic phenotype on mesenchymal cells, has subsequently been identified as a potent inhibitor of proliferation in most cells of epithelial origin. Our laboratory has investigated the role of specific second messengers in mediating the transcriptional responses of fibroblasts following addition of TGF-beta 1. Our studies indicate that TGF-beta 1, alone and in conjunction with epidermal growth factor (EGF), is capable of stimulating increases in both phosphoinositide metabolism and calcium influx, leading to significant increases in intracellular levels of Ca++ and inositol trisphosphate (IP3). Our data indicated that Ca++ influx and inositol phosphate release are coupled in Rat-1 cells, and suggested that influx of Ca++ from the extracellular medium is required for the change in IP3 accumulation observed in response to both EGF and TGF-beta 1. Using nuclear run-on analysis of the transcription of rat transin, a secreted metalloproteinase homologous to human stromelysin, we have also demonstrated a significant inhibition of transin transcription within 10 min of TGF-beta 1 treatment. The ability of TGF-beta 1 to inhibit transin gene transcription was not related to the TGF-beta 1-induced influx of Ca++ or to an increase in intracellular inositol phosphates, since inhibiting production of these second messengers failed to inhibit repression of the transin gene.

Stimulation of quiescent cultured fibroblasts with a variety of growth-promoting factors induces release of diacylglycerol (DG) and subsequent activation of protein kinase C (pkC), but the role of pkC in the induction of DNA synthesis and cell proliferation remains unclear. We have investigated the involvement of pkC in the response of Rat-1 fibroblasts to the newly described peptide endothelin-1 (Et-1), an agonist that is secreted by the vascular endothelium and that may play a role in the proliferative response of cells in the vessel wall. Addition of Et-1 to serum-deprived Rat-1 cells promoted DNA synthesis in the absence of additional factors and stimulated anchorage-independent growth in the presence of epidermal growth factor (EGF), indicating that Et-1 has many of the characteristics of a mitogen. The ability of Et-1 to stimulate both DNA synthesis and anchorage-independent growth was markedly reduced by the depletion of cellular pkC activity induced by prolonged exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, the ability of Et-1 to induce both second messenger production and transcription of c-fos and c-jun was largely independent of cellular pkC activity. Production of DG in response to Et-1 persisted for greater than 12 h and may account for the ability of Et-1 to augment the G1-S phase transition. Although these observations indicate that functional pkC is not an essential component of the proximal pathway leading to rapid changes in gene transcription and second messenger production in response to Et-1 treatment, the data suggest that activation of pkC is an essential component of the downstream events responsible for the stimulation of cell proliferation and anchorage-independent growth in Rat-1 cells exposed to Et-1.

Transin is a transformation-associated gene which is expressed constitutively in rat fibroblasts transformed by a variety of oncogenes and in malignant mouse skin carcinomas but not benign papillomas or normal skin. It has been demonstrated that, in nontransformed Rat-1 cells, transin RNA expression is modulated positively by epidermal growth factor (EGF) and negatively by transforming growth factor beta (TGF-beta); other peptide growth factors were found to have no effect on transin expression. Results presented here indicate that both protein synthesis and continuous occupancy of the EGF receptor by EGF were required for sustained induction of transin RNA. Treatment with TGF-beta inhibited the ability of EGF to induce transin, whether assayed at the transcriptional level by nuclear run-on analysis or at the level of transin RNA accumulation by Northern (RNA) blot analysis of cellular RNA. TGF-beta both blocked initial induction of transin transcription by EGF and halted established production of transin transcripts during prolonged treatment. These results suggest that TGF-beta acts at the transcriptional level to antagonize EGF-mediated induction of transin gene expression.

Serum-deprived human fibroblasts (HSWP cells) were loaded with either the fluorescent pH indicator 6-carboxy-4',5'-dimethylfluorescein or the calcium indicator quin 2, and the fluorescence of the intracellular probes was continuously monitored with a microspectrofluorometer. Addition of a cocktail of peptide growth factors causes intracellular alkalinization, which is blocked by amiloride or by replacement of extracellular Na+ with choline, confirming that mitogenic stimulation activates a Na+-H+ exchanger in HSWP cells. The exchanger is also activated by A23187, acid loading the cells, and stimulation of phospholipase activity with melittin. Growth factors and melittin activation of the exchanger have been demonstrated to be dependent on the mobilization of Ca2+ from intracellular stores. The intracellular alkalization and increase in Ca2+ activity due to both melittin and growth factors is inhibited by phospholipase inhibitors, indicating that phospholipase activity is necessary for the activation of the Na+-H+ exchanger and the mobilization of intracellular Ca2+.

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [3H]-thymidine. We have determined previously that pretreatment with the tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibits mitogen-stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen-stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen-stimulated inositol phosphate release occurs at 1-2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate-stimulated inositol phosphate release and Na influx. In addition, orthovanadate was determined to increase intracellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura-2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus-mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA-mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [3H]-thymidine.(ABSTRACT TRUNCATED AT 400 WORDS)

Transforming growth factor beta (TGF beta) alters the cellular response to epidermal growth factor (EGF) in a number of systems, but the underlying mechanisms for these alterations are largely unknown. We have examined second messenger formation in Rat-1 cells following treatment with EGF and/or TGF beta to determine whether the ability of TGF beta to potentiate some EGF-stimulated processes might be mediated by TGF beta-induced alterations in the signal transduction mechanism. Incubation of serum-deprived confluent Rat-1 cells with 10 ng/ml TGF beta resulted in a marked elevation of cellular inositol trisphosphate and inositol tetrakisphosphate levels, which were maximal at 4 h and maintained for at least 8 h. The effect of TGF beta on levels of inositol trisphosphate and inositol tetrakisphosphate was blocked by actinomycin D, suggesting that RNA synthesis was required for the TGF beta effect. While EGF stimulation induced a rapid and transient (5 min) rise in inositol phosphate levels in control cells, the EGF effect was considerably increased, both in magnitude and duration, by TGF beta treatment. Measurement of intracellular free Ca2+ with fura-2 demonstrated that TGF beta treatment markedly increased the EGF-stimulated rise in free Ca2+ and increased the duration of the response. The positive effects of TGF beta on EGF stimulation could not be explained on the basis of increased EGF binding to cells. We conclude that TGF beta treatment can both activate phosphatidylinositol turnover independently and also sensitize Rat-1 cells to stimulation by EGF.

Utilizing a digitonin-permeabilized cell system, we have studied the release of calcium from a non-mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (+/- 6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3-sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP-dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down-regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.

The mitogen-induced activation of Na+-H+ exchange was investigated in two cultured human fibroblast strains (HSWP and WI-38 cells) that, based on previous studies, differed in their response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (L. M. Vincentini and M. L. Villereal, Proc. Natl. Acad. Sci. USA 82: 8053-8056, 1985). The role of protein kinase C in the activation of Na+-H+ exchange was investigated by comparing the effects of TPA on Na+ influx, in vitro phosphorylation, and in vivo phosphorylation in both cell types. Although both cell types have significant quantities of protein kinase C activity that can be activated by TPA in intact cells, the addition of TPA to intact cells stimulates Na+ influx in WI-38 cells but not in HSWP cells, indicating that in HSWP cells the stimulation of protein kinase C is not sufficient to activate the Na+-H+ exchanger. Cells were then depleted of protein kinase C activity by chronic treatment with high doses of TPA. Both HSWP and WI-38 cells were rendered protein kinase C deficient by this treatment as determined by in vitro and in vivo phosphorylation studies. Protein kinase C-deficient HSWP cells lose the ability for TPA to inhibit the serum-induced activation of Na+-H+ exchange, but there is no reduction in the stimulation of Na+ influx by serum, bradykinin, vasopressin, melittin, or vanadate, indicating that protein kinase C activity is not necessary for the mitogen-induced activation of Na+-H+ exchange in HSWP cells by agents known to stimulate phosphatidylinositol turnover (G. A. Jamieson and M. Villereal. Arch. Biochem. Biophys. 252: 478-486, 1987). In contrast, depletion of protein kinase C activity in WI-38 cells significantly reduces both the TPA- and the serum-induced activation of the Na+-H+ exchange system, suggesting that protein kinase C activity is necessary for at least a portion of the mitogen-induced activation of the Na+-H+ exchanger in WI-38 cells. These results indicate that the mechanisms for regulating Na+-H+ exchange can differ dramatically between different types of fibroblasts.

Transforming growth factor type beta (TGF beta) alters the cellular response to epidermal growth factor (EGF) for a variety of processes ranging from early transport activities and gene transcription to mitogenesis. In order to test the hypothesis that altered signal transduction mechanisms may mediate both the transforming effects of TGF beta and the modulation of EGF-stimulated processes by TGF beta, we have examined second messenger levels in response to growth factor treatment. The addition of EGF or prolonged treatment with TGF beta increased the rate of 45Ca influx in serum-deprived, confluent Rat-1 cells, while the addition of EGF to TGF beta-pretreated cells produced an additive increase in Ca2+ influx. The stimulation of Ca2+ influx by TGF beta was only observed at incubation times greater than 1 h and was inhibited by inclusion of actinomycin D, suggesting that a newly transcribed gene product was required for the observed response to TGF beta. Both EGF and TGF beta displayed similar time and concentration dependencies for stimulation of Ca2+ influx and for accumulation of inositol trisphosphate (IP3). The increase in IP3 accumulation in response to either EGF or TGF beta required the presence of extracellular Ca2+, and the observed concentration dependencies were similar for the stimulation of phosphatidylinositol turnover and Ca2+ influx. The EGF- and TGF beta-stimulated increases in Ca2+ influx could be blocked by cobalt, cadmium, and [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, but not by specific Ca2+ channel blockers such as nifedipine or verapamil, suggesting that these growth factors do not act via L-type voltage-sensitive calcium channels. Those calcium blockers which inhibited Ca2+ influx also inhibited inositol phosphate release. These data, taken together, indicate that Ca2+ influx and inositol phosphate release are coupled in Rat-1 cells and suggest that influx of Ca2+ from the extracellular medium is responsible for the changes in IP3 accumulation observed in response to both EGF and TGF beta.

Growth-arrested human fibroblasts respond to mitogenic stimulation with a rapid, transient increase in cytoplasmic free Ca2+. This event may be crucial to the activation of Na/H exchange and subsequent DNA synthesis. Previous studies have implicated calmodulin (CaM) as a possible mediator of the effects of Ca2+ on these processes. here, we demonstrate that a specific CaM-dependent protein kinase (CaM-PK) system is rapidly activated in quiescent fibroblasts stimulated by a variety of mitogens. Cytoplasmic extracts of two human fibroblast cell types contained a major Ca2+-stimulated phosphoprotein of Mr 100,000 and pI 6.8 (Mr 100,000). This protein was shown by peptide mapping and immunological criteria to be identical to the prominent CaM-PK III substrate previously identified in a number of mammalian cells and tissues (Palfrey, H. C. (1983) FEBS Lett. 157, 183-190; Nairn, A.C., Bhagat, B., and Palfrey, H.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7939-7943). Stimulation of 32P-labeled serum-deprived fibroblasts with serum, individual growth factors (bradykinin, vasopressin, and epidermal growth factor), or Ca2+ ionophores resulted in a rapid 2- to 10-fold increase in the phosphorylation of Mr 100,000 as determined by immunoprecipitation using polyclonal antibodies. With serum or individual growth factors, the effect peaked at 0.5-1 min then declined back to base line within 5 min. Time course studies showed that the phosphorylation state of Mr 100,000 closely paralleled but lagged slightly behind the Ca2+ transient (measured with fura-2). Thus, dephosphorylation of Mr 100,000 must follow shortly after Ca2+ levels begin to decline. The effects of serum, bradykinin, and vasopressin on both the rise in intracellular Ca2+ and the phosphorylation of Mr 100,000 were independent of external Ca2+, whereas the effects of epidermal growth factor and A23187 required external Ca2+. Phosphorylation of Mr 100,000 in intact cells took place on threonine residues, a major portion occurring in the same major phosphopeptide found in the protein labeled in vitro. These results show that mitogenic activation of human fibroblasts leads to the binding of Ca2+ to CaM and the subsequent activation of CaM-dependent processes.