Urinary transforming growth factors in neoplasia: separation of 125I-labeled transforming growth factor-alpha from epidermal growth factor in human urine.

Purified human epidermal growth factor (hEGF) from urine promotes anchorage-independent cell growth in soft agar medium. This growth is enhanced by transforming growth factor-beta (TGF-beta), and is specifically inhibited by hEGF antiserum. Transforming growth factors of the alpha type (TGF-alpha), potentially present in normal human urine or urine from tumor-bearing patients, also promote anchorage-independent cell growth and compete with EGF for membrane receptor binding. Consequently, TGF-alpha cannot be distinguished from urinary hEGF by these two functional assays. Therefore, a technique for separation of TGF-alpha and related peptides from urinary EGF based on biochemical characteristics would be useful. Radioiodination of characterized growth factors [mouse EGF (mEGF), hEGF, and rat TGF-alpha (rTGF-alpha)], which were then separately added to human urine, was used to evaluate a resolution scheme that separates TGF-alpha from the high level of background hEGF present in human urine. Commercially available methyl bonded microparticulate silica efficiently adsorbed the 125I-labeled mEGF, 125I-labeled hEGF, and 125I-labeled rTGF-alpha that were added to 24-h human urine samples. Fractional elution with acetonitrile (MeCN) of the adsorbed silica released approximately 70 to 80% of the 125I-labeled mEGF and 125I-labeled hEGF between 25 and 30% MeCN, and over 80% of the 125I-labeled rTGF-alpha between 15 and 25% MeCN, with retention after dialysis of less than 0.2 and 1.7% of the original urinary protein, respectively. Consequently, a single-step enrichment of about 400-fold for mEGF and hEGF, and 50-fold for rTGF-alpha were achieved rapidly. Subsequent Bio-Gel P-10 chromatography indicated that 125I-labeled mEGF and 125I-labeled hEGF eluted later than would be predicted on the basis of their reported molecular weight of approximately 6000, whereas 125I-labeled rTGF-alpha eluted from Bio-Gel P-10 at an approximate molecular weight of 8000 to 9000. 125I-labeled rTGF-alpha bound to carboxymethyl cellulose and eluted at a less acidic pH than did hEGF. On reverse phase high performance liquid chromatography using a linear gradient of 18 to 35% MeCN over 120 min, 125I-labeled rTGF-alpha was comparatively hydrophilic, eluting at 22% MeCN in contrast to the more hydrophobic 125I-labeled hEGF, which eluted at 27% MeCN. These observed biochemical differences between hEGF and TGF-alpha provide a rationale for resolution of these and perhaps other related putative transforming growth factors from bulk urine of tumor-bearing patients.

Variant forms of rat epidermal growth factor present in the urine of nude rats bearing human tumors.

Epidermal growth factor (EGF) receptor-binding peptides from the urine of tumor patients have been reported to differ in molecular weight and relative hydrophobicity from those of normal individuals. Nude rats bearing human large cell lung carcinomas or chondrosarcomas and non-tumor-bearing sibling control rats were used to investigate the contributions of tumor and host to urinary EGF-related peptide growth factors. Peptides were adsorbed from urine onto methyl-bonded silica and eluted according to their relative hydrophobicity by a stepwise gradient of aqueous acetonitrile. Total EGF receptor-binding activity relative to urinary creatine was elevated in the urine of only one group of tumor-bearing rats. However, the proportion of relatively hydrophilic activity was increased markedly in all three groups of tumor-bearing rats. Rats bearing a large cell lung cancer excreted unusually hydrophilic Mr 6000 peptides that were chromatographically similar to transforming growth factor alpha on reverse phase high performance liquid chromatography but proved to be rat EGF by radioimmunoassay (RIA). EGF receptor-binding activity that was common to the urine of tumor-bearing animals regardless of tumor type, but more hydrophilic than that from control rats, had Mr 60,000, 30,000, 12,000, and 4,000 to 7,000 components. All reacted fully in the rat EGF RIA and were negative for human EGF and transforming growth factor alpha by RIA. A more hydrophobic fraction of EGF receptor-binding activity, common to control and tumor-bearing animals, contained Mr 33,000, 5,000 to 7,000, and 2,000 to 5,000 components. High performance liquid chromatography and gel electrophoresis of the Mr 33,000 activity revealed a high molecular weight rat EGF comparable to that reported in human urine. No human EGF or transforming growth factor alpha was detected by RIA in any of the active fractions from tumor-bearing rat urine. Thus, all EGF receptor-binding activity appeared to derive from rat EGF produced by the rat host and not by the xenografted tumors.

Crystal structure of a carcinogen:nucleoside adduct.

The product of reaction between the carcinogen, 7-bromomethyl-12-methylbenz[a]anthracene, and 2'-deoxyadenosine, i.e., N6-(12-methylbenz[a]anthracenyl-7-methyl)deoxyadenosine, has been prepared and characterized, and its structure has been determined by X-ray crystallographic techniques. The major structural features are: (a) the adenine and polycyclic aromatic hydrocarbon residues lie nearly perpendicular to one another; (b) the conformation about the glycosidic bond is syn, rather than anti, and an internal hydrogen bond between the deoxyribose 5'-hydroxyl group and N(3) of the adenine residue is present; and (c) the more planar anthracene portion of the hydrocarbon is stacked between adenine residues of other molecules throughout the crystal.

Selective activity of phenylacetate against malignant gliomas: resemblance to fetal brain damage in phenylketonuria.

Phenylacetate, a deaminated metabolite of phenylalanine, has been implicated in damage to immature brain in phenylketonuria. Because primary brain tumors are highly reminiscent of the immature central nervous system, these neoplasms should be equally vulnerable. We show here that sodium phenylacetate can induce cytostasis and reversal of malignant properties of cultured human glioblastoma cells, when used at pharmacological concentrations that are well tolerated by children and adults. Treated tumor cells exhibited biochemical alterations similar to those observed in phenylketonuria-like conditions, including selective decline in de novo cholesterol synthesis from mevalonate. Because gliomas, but not mature normal brain cells, are highly dependent on mevalonate for production of sterols and isoprenoids vital for cell growth, sodium phenylacetate would be expected to affect tumor growth in vivo while sparing normal tissues. Systemic treatment of rats bearing intracranial gliomas resulted in significant tumor suppression with no apparent toxicity to the host. The data indicate that phenylacetate, acting through inhibition of protein prenylation and other mechanisms, may offer a safe and effective novel approach to treatment of malignant gliomas and perhaps other neoplasms as well.

Human brain tumor-associated urinary high molecular weight transforming growth factor: a high molecular weight form of epidermal growth factor.

Urinary protein obtained from a patient with a highly malignant brain tumor (astrocytoma, grade IV) was adsorbed to trimethylsilyl controlled-pore glass beads and selectively eluted with acetonitrile to yield a high molecular weight (HMW) human transforming growth factor (hTGF). This HMW hTGF promoted clonogenic cell growth in soft agar and competed for membrane receptors with mouse epidermal growth factor. After surgical resection of the tumor, no HMW hTGF was found in urine. HMW hTGF generated a human EGF (hEGF) radioimmunoassay competitive binding curve similar to that of hEGF and parallel to that of a highly purified HMW form of hEGF previously reported to be present in trace concentrations in normal human urine. Both hEGF and HMW hEGF were clonogenic in soft agar, and their clonogenic activity as well as that of HMW hTGF was inhibited by anti-hEGF serum. Both HMW hTGF and HMW hEGF had 20 to 25% of the radioreceptor binding activity of hEGF. HMW hTGF purified from the pooled urine of several patients with malignant astrocytomas and HMW hEGF purified from normal control urine comigrated at Mr 33,000. Thus, HMW hTGF was indistinguishable from HMW hEGF in terms of apparent molecular size, epidermal growth factor receptor binding activity, epidermal growth factor immunoreactivity, and clonogenic activity. Urinary HMW hEGF/hTGF may be of tumor cell origin or may represent a response of normal host tissues to the tumor or its products.

Activation of the cholesterol pathway and Ras maturation in response to stress.

All cells depend on sterols and isoprenoids derived from mevalonate (MVA) for growth, differentiation, and maintenance of homeostatic functions. In plants, environmental insults like heat and sunlight trigger the synthesis of isoprene, also derived from MVA, and this phenomenon has been associated with enhanced tolerance to heat. Here, we show that in human prostate adenocarcinoma PC-3M cells heat shock leads to activation of the MVA pathway. This is characterized by a dose- and time-dependent elevation in 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) activity, enhanced sterol and isoprenoid synthesis, and increased protein prenylation. Furthermore, prenylation and subsequent membrane localization of Ras, a central player in cell signaling, was rapidly induced following heat stress. These effects were dose-dependent, augmented with repeated insults, and were prevented by culturing cells in the presence of lovastatin, a competitive inhibitor of HMGR. Enhanced Ras maturation by heat stress was also associated with a heightened activation of extracellular signal-regulated kinase (ERK), a key mediator of both mitogenic and stress signaling pathways, in response to subsequent growth factor stimulation. Thus, activation of the MVA pathway may constitute an important adaptive host response to stress, and have significant implications to carcinogenesis.

Glycosylated insulin-like growth factor II promoted expansion of granulocyte-macrophage colony-forming cells in serum-deprived liquid cultures of human peripheral blood cells.

This report presents the results of studies investigating the effect of a glycosylated form of insulin-like growth factor II with an apparent molecular weight of 15,000 (appM(r) = 15K IGF-II) and one with a molecular weight of 7500 (M(r) = 7.5K IGF-II) on the expansion of granulocyte-macrophage colony-forming cells (GM-CFC) in human peripheral blood cells. Blood cells were enriched for GM-CFC and other CFCs, and liquid cultures of these cells were established in serum-deprived medium supplemented with either interleukin-3 (IL-3) alone (no insulin or IGF added to the medium) or with IL-3 plus M(r) = 7.5K recombinant (r) IGF-II or appM(r) = 15K IGF-II. After incubation for 3 days or 1 week, the blood cells were subcultured in plasma clots, and the number of colonies detected at 7 days (D7) and at 14 days (D14) was used to calculate the number of D7 GM-CFC and D14 GM-CFC. The number of GM-CFC in liquid cultures of blood cells incubated for 3 days with IL-3 alone was similar to the number added at day 0. By 1 week, the number of D14 GM-CFC and D7 GM-CFC had increased to 3.5 +/- 0.9-fold (p = .03) and two- to 50-fold (p = .04) of the number at day 3, respectively. There were 1.5- to six-fold more D7 GM-CFC in cultures of blood cells incubated for 1 week with IL-3 plus either 100 ng/mL M(r) = 7.5K IGF-II or 200 ng/mL appM(r) = 15K IGF-II than after incubation with IL-3 alone. appM(r) = 15K IGF-II also promoted a two-fold increase in the number of D14 GM-CFC. appM(r) = 15K IGF-II promoted a greater increase in D14 GM-CFC than incubation with IL-3 alone even for blood samples in which M(r) = 7.5K IGF-II did not promote such an increase. The results of these studies demonstrate that physiologic concentrations of appM(r) = 15K IGF-II and M(r) = 7.5K IGF-II increased the number of GM-CFC more than IL-3 alone and suggest that appM(r) = 15K IGF-II was more potent than M(r) = 7.5K IGF-II in augmenting IL-3-induced expansion of GM-CFC in serum-deprived liquid cultures of peripheral blood cells.

Differentiation of cultured human melanoma cells induced by the aromatic fatty acids phenylacetate and phenylbutyrate.

The increasing incidence of melanoma and the poor responsiveness of disseminated disease to conventional treatments call for the development of new therapeutic approaches. Phenylacetate, a nontoxic differentiation inducer, can suppress the growth of other neuroectodermal tumors, i.e., gliomas, in laboratory models and in humans. This finding led us to explore the efficacy of phenylacetate and related aromatic fatty acids in melanoma. Phenylacetate and phenylbutyrate were found to a) induce selective cytostasis and maturation of cultured human melanoma cells, b) modulate the expression of genes implicated in tumor metastasis (type IV collagenase and tissue inhibitor of metalloproteinases-2) and immunogenicity (HLA class I); and c) enhance the efficacy of other agents of clinical interest, including retinoids, interferon-alpha, suramin, and 5-aza-2'-deoxycytidine. Reflecting on the phenotypic heterogeneity of melanoma, the degree of biologic alterations induced by phenylacetate/phenylbutyrate varied significantly among the tumor cell lines tested. Although losing invasive capacity and tumorigenicity in athymic mice, poorly differentiated cells exhibited only a marginal change in morphology, remained amelanotic, and resumed growth after treatment was discontinued. By contrast, treatment of melanoma cells that were in a more advanced stage of maturation resulted in profound alterations in cell growth, morphology, and pigmentation consistent with terminal differentiation. The in vitro antitumor activity was observed with nontoxic, pharmacologic concentrations of phenylacetate and phenylbutyrate, suggesting potential clinical use of these drugs in the treatment of melanomas.

A multi-institutional experience with stereotactic radiosurgery for solitary brain metastasis.

PURPOSE: A multi-institutional experience in radiosurgery for solitary brain metastases was combined to identify factors associated with safety, efficacy, tumor control, and survival. MATERIALS AND METHODS: A review of 116 patients with solitary brain metastases who underwent gamma knife stereotactic radiosurgery at five institutions was performed. The median follow-up was 7 months following radiosurgery and 12 months following diagnosis. Minimum tumor doses varied from 8-30 Gy (mean, 17.5 Gy). Forty-five patients failed prior radiotherapy and 71 had no prior brain irradiation. Fifty-one patients had radiosurgery alone and 65 underwent combined radiosurgery with fractionated large-field radiotherapy (mean dose, 33.8 Gy). RESULTS: Median survival was 11 months after radiosurgery and 20 months after diagnosis. Follow-up documented local tumor control in 99 patients (85%), tumor recurrence in 17 (15%), and documented radiation necrosis in one (1%). The 2-year actuarial tumor control rate was 67 +/- 8%. Tumor histology affected survival (better for breast cancer, p = .004) and local control (better for melanoma and renal cell, p = .0003) in multivariate analyses. Combined fractionated radiotherapy and radiosurgery improved local control (p = 0.111), but not survival in multivariate testing. CONCLUSION: Radiosurgery is effective in controlling solitary brain metastases with low morbidity. Further study is needed to better define optimum treatment parameters for radiosurgery.

Gamma knife for glioma: selection factors and survival.

PURPOSE: To determine factors associated with survival differences in patients treated with radiosurgery for glioma. METHODS AND MATERIALS: We analyzed 189 patients treated with Gamma Knife radiosurgery for primary or recurrent glioma World Health Organization (WHO) Grades 1-4. CONCLUSION: The median minimum tumor dose was 16 Gy (8-30 Gy) and the median tumor volume was 5.9 cc (1.3-52 cc). Brachytherapy selection criteria were satisfied in 65% of patients. Median follow-up of all surviving patients was 65 weeks after radiosurgery. For primary glioblastoma patients, median survival from the date of pathologic diagnosis was 86 weeks if brachytherapy criteria were satisfied and 40 weeks if they were not (p = 0.01), indicating that selection factors strongly influence survival. Multivariate analysis showed that increased survival was associated with five variables: lower pathologic grade, younger age, increased Karnofsky performance status (KPS), smaller tumor volume, and unifocal tumor. Survival was not found to be significantly related to radiosurgical technical parameters (dose, number of isocenters, prescription isodose percent, inhomogeneity) or extent of preradiosurgery surgery. We developed a hazard ratio model that is independent of the technical details of radiosurgery and applied it to reported radiosurgery and brachytherapy series, demonstrating a significant correlation between survival and hazard ratio. CONCLUSIONS: Survival after radiosurgery for glioma is strongly related to five selection variables. Much of the variation in survival reported in previous series can be attributed to differences in distributions of these variables. These variables should be considered in selecting patients for radiosurgery and in the design of future studies.

Cytostatic activity of phenylacetate and derivatives against tumor cells. Correlation with lipophilicity and inhibition of protein prenylation.

The aromatic fatty acid phenylacetate, a common metabolite of phenylalanine, shows promise as a relatively non-toxic drug for cancer treatment. This slowly metabolized fatty acid alters tumor cell lipid metabolism causing, among other effects, inhibition of protein prenylation critical to malignant growth. In pursuit of more potent analogues, we have examined the activity of related compounds against tumor cell lines established from patients with advanced prostatic carcinoma, glioblastomas, and malignant melanoma. Like phenylacetate, derivatives containing alpha-carbon or ring substitutions induced cytostasis and phenotypic reversion at non-toxic concentrations. Potency was correlated with the degree of calculated lipophilicity of the aromatic fatty acid, and the extent of inhibition of protein prenylation. Remarkably, a parallel cytostatic activity was reported in embryonic plant cells, which respond to phenylacetate and its analogues in the same concentration range and the same rank order of lipophilicity. These data suggest that phenylacetate and its analogues may act through common mechanisms to inhibit the growth of vastly divergent, undifferentiated cell types, and provide a basis for the development of new agents for the treatment of human malignancies.

Transcriptional upregulation of gamma-globin by phenylbutyrate and analogous aromatic fatty acids.

Phenylbutyrate has been shown recently to induce fetal hemoglobin (HbF) production in patients with sickle cell anemia and beta thalassemia. We have now examined related aromatic fatty acids in order to define the range of active structures and identify plausible mechanisms of action. Structure-function analysis revealed that for effective stimulation of HbF in erythroid precursors: (1) the ideal length for the aliphatic side chain is four carbons; (2) oxygen or sulfur substitutions in the carboxylic chain are allowed, as evidenced by the equal or increased activity of phenoxypropionate, benzylthioglycolate, and benzyloxyacetate compared with phenylbutyrate; and (3) blocking the carboxylate group by conversion to the amide form greatly reduces potency. Molecular analysis indicated that the prototype agent, phenylbutyrate, increases HbF production through transcriptional activation of the gamma-globin gene. The latter contains a butyrate responsive promoter known to up-regulate transcription in the presence of short-chain fatty acids of three to five carbons. To determine whether stimulation of an element in this promoter by phenylbutyrate and its analogues might contribute to their mechanism of action, we used a transient expression system involving K562 erythroleukemia cells transfected with a luciferase reporter gene driven by the minimum gamma-globin promoter. Transcriptional activation in this experimental system correlated well with the capacity of an aromatic fatty acid to increase HbF production in erythroid precursors (r = 0.94). Our studies identify potent analogues of phenylbutyrate for the treatment of beta-chain hemoglobinopathies, and suggest that stimulation of a butyrate responsive promoter may be responsible for their activity.

Activation of a human peroxisome proliferator-activated receptor by the antitumor agent phenylacetate and its analogs.

The aromatic fatty acid phenylacetate and its analogs induce tumor cytostasis and differentiation in experimental models. Although the underlying mechanisms of action are not clear, effects on lipid metabolism are evident. We have now examined whether these compounds, structurally similar to the peroxisome proliferator clofibrate, affect the human peroxisome proliferator-activated receptor (hPPAR), a homolog of the rodent PPAR alpha, a transcriptional factor regulating lipid metabolism and cell growth. Gene transfer experiments showed activation of hPPAR, evident by the increased expression of the reporter gene chloramphenicol acetyltransferase linked to PPAR-response element from either the rat acyl-CoA oxidase or rabbit CYP4A6 genes. The relative potency of tested drugs in the co-transfection assay was: 4-iodophenylbutyrate > 4-chlorophenylbutyrate > clofibrate > phenylbutyrate > naphthylacetate > 2,4-D > 4-chlorophenylacetate > phenylacetate >> indoleacetate. Phenylacetylglutamine, in which the carboxylic acid is blocked, was inactive. The ability of the aromatic fatty acids to activate PPAR was confirmed in vivo, as CYP4A mRNA levels increased in hepatocytes of treated rats. Further studies using human prostate carcinoma, melanoma, and glioblastoma cell lines showed a tight correlation between drug-induced cytostasis, increased expression of the endogenous hPPAR, and receptor activation documented in the gene-transfer model. These results identify phenylacetate and its analogs as a new class of aromatic fatty acids capable of activating hPPAR, and suggest that this nuclear receptor may mediate tumor cytostasis induced by these drugs.

Patients' attitude about outcomes and the role of gamma knife radiosurgery in the treatment of vestibular schwannomas.

In one strategy for the treatment of unilateral vestibular schwannomas measuring up to 3 cm in diameter, decision analysis shows that gamma knife radiosurgery has probabilistic dominance over microsurgical resection. That is, radiosurgery produces better results for any value assigned to treatment outcomes (ranked from best to worst) of the following: no complications, hearing loss only, residual/recurrent tumor, facial paralysis, major disability, or death. This little-known principle of decision analysis will be explained. It implies that when patients prefer the preservation of facial nerve function, even if that requires leaving a tumor remnant, then gamma knife radiosurgery is a better treatment strategy than microsurgical resection.

Exposure of two interspaces for lumbar disc surgery.

In a study of matched pairs of patients with a single ruptured disc, exploration of an additional lumbar interspace did not increase the morbidity of surgery. The author believes that the desire to avoid additional surgery does not, by itself, justify routine myelography.

Fluorescence of hydrocarbon-deoxyribonucleoside adducts.

In comparison with the fluorescence emission spectra of 7-methylbenz[a]-anthracene-nucleoside adducts, the fluorescence emission spectra of hydrocarbon-deoxyribonucleoside adducts containing a methyl substituent in the "bay region" lack spectral resolution at room temperature and appear at substantially longer wavelength. This spectral resolution is improved when spectra are measured at 77 K and an irreversible spectral shift to shorter wavelength, accompanied by improved resolution, results from mild acid hydrolysis. These spectral properties peculiar to the "bay region-substituted" adducts presumably result from an intramolecular interaction between the hydrocarbon fluorophore and the attached nucleoside brought about, in the examples studied here, by the presence of the 12-methyl group in 7,12-dimethylbenz[awanthracene (DMBA) and in 7-hydroxymethyl-12-methylbenz[a]anthracene. This interaction suggests that the site of nucleoside attachment is in close proximity to the 12-methyl group and that binding occurs, therefore, through the intermediacy of a 3,4-diol-1,2-oxide, i.e. a "bay region" diol-epoxide in each case.

Computer-aided diagnosis of lumbar disc herniation.

A microcomputer was programmed to accept data on the history and physical findings of patients, with low-back pain, suspected of having a herniated lumbar intervertebral disc, then suggest a likely diagnosis, with probability, and make suggestions for further management. Formal decision analytic techniques were used to test for the threshold of diagnostic likelihood that would make the expected value of laminotomy for excision of a herniated disc greater than the expected value of non-surgical management. The program is recursive, using its results to update its data base, and become more "intelligent." In a blinded evaluation, an expert could not detect a significant difference between the output of the computer and the diagnoses and treatment plans of ten clinicians.

Phenylacetate and phenylbutyrate as novel, nontoxic differentiation inducers.

Phenylacetate and analogs represent a new class of pleiotropic growth regulators that alter tumor cell biology by affecting gene expression at both the transcriptional and post transcriptional levels. Based on these findings, NaPA and NaPB entered clinical trials at the National Cancer Institute. Ongoing phase I studies with NaPA, involving adults with prostate and brain cancer, have confirmed that therapeutic levels can be achieved with no significant toxicities, and provide preliminary evidence for benefit to patients with advanced disease (Thibault et al., submitted).

Gamma knife radiosurgery: brain surgery without an incision.

Radiosurgery is the precise targeting of ionizing radiation to inactivate or destroy pathologic tissue while sparing adjacent tissue. Like surgery, radiosurgery is done in a single treatment; unlike surgery, radiosurgery does not require an anesthetic or an incision. A specific device for neurosurgery, the gamma knife, uses 201 separate 60Co sources to crossfire gamma rays through a collimator helmet across an intracranial target. This device has been used to treat more than 200 patients at Presbyterian Hospital of Dallas since December 1989. Most patients had either inoperable lesions or residual/recurrent lesions after craniotomy. No patient died and only one developed clinical radionecrosis. Only an overnight hospital stay was required, and patients could resume previous work and activities the day after treatment. Early results parallel reported outcomes in patients treated in Sweden, England, and elsewhere in the United States. In selected patients, the gamma knife is an effective, low-risk, and cost-effective alternative to conventional neurosurgery.