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Object: The treatment of solitary vestibular schwannomas by performing Gamma Knife surgery is well established. It has been reported that decreasing the surface dose reduces patient morbidity, especially facial weakness and numbness. The authors of this retrospective study examine patient data from a single center to determine if low-dose (< or = 14 Gy) GKS controls tumor growth as effectively as higher doses (> 14 Gy)., Methods: Based on the formula for ellipsoid volumes, the tumor volumes were calculated using measurements from MR images obtained at follow up in patients treated at the authors' center. Follow-up data were available in 159 patients with a mean age of 59.5 +/- 14.2 years at treatment. Fifty-six percent of the patients were women and 53.5% of the tumors were located on the right side of the brain. The mean tumor volume was 3.3 +/- 4.3 cm3 with 10% of the tumors having volumes larger than 8 cm3. After GKS, smaller tumors (> or = 40% decrease in volume) were observed in 44.8% of patients treated with a low dose and in 48.8% treated with a high dose. Enlarged tumors (> or = 40% increase in volume) were seen in 5.2% of the patients receiving a low dose and 2.3% of those receiving a high dose. These differences were not statistically significant. Patients who had been followed up for longer than 5 years after treatment had median residual volumes of only 28.2% of the starting volume in the low-dose group and 26% in the high-dose group. This difference was statistically not significant., Conclusions: No statistically significant differences were observed between tumors given low-dose radiation treatment and those given high-dose radiation treatment.

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.

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).

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.

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.

Clinical and imaging results of Gamma Knife treatment of 100 consecutive patients with intracranial meningiomas are reported. Only 1 patient refused follow-up imaging and her symptoms remain improved after 1 year. Mean values for the patient and treatment parameters were age 61 years, duration of symptoms 3.6 years, time since diagnosis 3 years, average tumor diameter 2.4 cm, surface radiation dose 15 Gy and number of isocenters 5. Clinical outcomes revealed that 6 were improved, 75 were unchanged and 17 had deteriorated. Of the latter, 8 were operated, 4 were treated medically and 5 died. Imaging follow-up showed no growth in 87 patients. The size of tumors treated ranged from 0.66 to 6.8 cm average diameter. In the 77 patients with tumors with an average diameter of 3 cm or less, only 2 (3%) showed further growth, and none died of tumor-related causes.

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.

Cinnamic acid, a naturally occurring aromatic fatty acid of low toxicity, has a long history of human exposure. We now show that cinnamic acid induces cytostasis and a reversal of malignant properties of human tumor cells in vitro. The concentration causing a 50% reduction of cell proliferation (IC50) ranged from 1 to 4.5 mM in glioblastoma, melanoma, prostate and lung carcinoma cells. Using melanoma cells as a model, we found that cinnamic acid induces cell differentiation as evidenced by morphological changes and increased melanin production. Moreover, treated cells had reduced invasive capacity associated with modulation of expression of genes implicated in tumor metastasis (collagenase type IV, and tissue inhibitor metalloproteinase 2) and immunogenicity (HLA-A3, class-I major histocompatibility antigen). Further molecular analysis indicated that the anti-tumor activity of cinnamic acid may be due in part to the inhibition of protein isoprenylation known to block mitogenic signal transduction. The results presented here identify cinnamic acid as a new member of the aromatic fatty acid class of differentiation-inducers with potential use in cancer intervention.

The aromatic fatty acids phenylacetate (PA) and phenylbutyrate (PB) induce tumour cell differentiation in experimental models and both are currently in clinical trials. The purpose of this study was to determine the effect of these antitumour agents on the expression of transforming growth factor-alpha (TGF-alpha) in neoplastic cells. Treatment of human melanoma 1011 cultures with either PA or PB caused over 40-fold increase in TGF-alpha biosynthesis and secretion into the media. Whereas elevation in TGF-alpha mRNA steady-state levels became evident within 6-12 h and reached peak quantities the following day, the amounts of its coded protein increased gradually over a period of 5 days of treatment. Further molecular analysis revealed that regulation of TGF-alpha expression occurred at the transcriptional level. In contrast to TGF-alpha, expression of its receptor remained below detectable levels, indicating that an autocrine loop involving this growth factor is unlikely. Interestingly, the increase in TGF-alpha production paralleled drug-induced cytostasis and differentiation defined by morphological changes and increased melanogenesis. Like PA and PB, other differentiation inducers such as all-trans-retinoic acid, dimethyl sulfoxide, and 5-aza-2'-deoxycytidine, all induced TGF-alpha expression in the melanoma cells. The close association between enhanced TGF-alpha production and melanoma cell differentiation suggests that this growth factor, often linked to mitogenesis, may play a novel role in tumour differentiation by PA and PB.

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.

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.

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.

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.

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.

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.

Decision analysis uses a tree-like diagram to describe treatment outcomes, the likelihood and value of which are obtained from the literature. A personal computer program entitled 'D-Tree' was used to compare radiosurgery to both microsurgery and nonoperative management of cerebral arteriovenous malformations (AVMs). The results indicated that radiosurgery is the treatment of choice for all AVMs up to 3 cm in diameter, not just those considered inoperable. Sensitivity analysis, to show the effect on treatment preference of a range of assumptions concerning outcome probabilities and values, suggested that for microsurgery to provide more years of life expectancy than radiosurgery, the obliteration rate of radiosurgery should be less than 60%. If the radiosurgical obliteration rate is 85%, then microsurgery must have a zero mortality and morbidity rate to equal the results of radiosurgery.

A procedure that combined ion exchange, gel permeation, and insulin-like growth factor-binding protein 3 (IGF-BP-3) affinity chromatography with chromatofocusing and reversed-phase high pressure liquid chromatography was used to isolate high molecular weight precursors of human insulin-like growth factor II (IGF-II) from acetic acid extracts of Cohn fraction IV1. Two precursors had isoelectric points (pI) of 5.1 and 5.4 and apparent Mr values of 15,000 and 11,500, respectively. An apparent Mr = 16,000 RLPG/Ser29 variant of IGF-II was also identified in the acetic acid extracts. Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent Mr = 15,000 IGF-II (pI 5.1), following its digestion with the endoprotease Lys-C, indicated the carboxyl terminus of this precursor was near or at Lys88. During the sequencing of this peptide, a sharply reduced yield of derivatized amino acid occurred at cycle 10, indicating that Thr75 had been posttranslationally modified, possibly by O-glycosylation. To evaluate this possibility, the 125I-labeled high molecular weight IGF-IIs and their endoprotease-generated peptides were treated with glycosidases, and their effects were determined from the change in relative mobilities of the polypeptide and peptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neuraminidase treatment of apparent Mr = 15,000 and 11,500 IGF-II reduced their Mr values to a common value of 10,500. When the desialylated precursors of IGF-II were treated with O-glycosidase, but not when treated with N-glycosidase, the Mr values were reduced further to about 10,000. This was the Mr value that would be predicted for an unglycosylated form of precursor IGF-II that had a carboxyl-terminal end at or near Lys88. When the Ser66-Lys88 endoprotease-generated E domain peptides from pI5.1 and 5.4 high Mr IGF-II were treated with the glycosidases, they had relative mobility changes during sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were similar to those of the intact precursors. Finally, the association of O-linked oligosaccharide with the E domain peptide of IGF-II was confirmed by demonstrating the specificity of binding of the Ser66-Lys88 asialoglycopeptide to jackfruit lectin.

In an effort to explore the use of polypeptide growth factors as potential markers for cancer detection, we have identified the presence of transforming growth factor-alpha (TGF-alpha) in pooled urine of patients with metastatic breast cancer by a commercial radioimmunoassay (RIA) based on a rabbit antiserum raised to the C-terminal 17aa synthetic fragment of rat TGF-alpha. This TGF-alpha RIA detected both high molecular weight (HMW) and low molecular weight (LMW) forms of TGF-alpha in the conditioned media of a breast cancer cell line (MDA-MB-231) and in the urine of healthy women and those with breast cancer. The ratio of HMW to LMW species of TGF-alpha by RIA after Bio-Gel P-100 chromatography was approximately equal in pooled urine samples from both healthy women and those with breast cancer, and in the conditioned media from the cell line MDA-MB-231. Using established procedures for concentrating urinary proteins from 24-h urine samples by adsorption onto methyl-bonded microparticulate silica and selective elution by acetonitrile, TGF-alpha RIA results from women with disseminated breast carcinoma were compared with those of healthy pre- and post-menopausal control women. Analysis indicated a median TGF-alpha value of 981 ng/g urinary creatinine for urine samples from cancer patients (range 608 to 1737) and 642 ng/g creatinine (range 417 to 941) for control urine samples. Although the difference was statistically significant (p less than 0.05), urinary TGF-alpha detection with this assay method appears to have limited usefulness as a diagnostic marker for metastatic human adenocarcinoma of the breast.(ABSTRACT TRUNCATED AT 250 WORDS)

A tumor-associated growth factor was identified in a 22-liter pool of urine from patients with disseminated breast cancer using an isolation scheme which separates transforming growth factor-alpha (TGF-alpha) from the high level of human epidermal growth factor (hEGF) normally present in urine. Concentration of urinary proteins by adsorption onto methyl bonded microparticulate silica, selective elution by acetonitrile, and subsequent gel permeation, cation exchange, and high performance liquid chromatography, resolved an EGF-related growth factor which generated a competitive binding curve similar to that of synthetic rat TGF-alpha in radioimmunoassay. A 26-liter pool of urine from normal subjects, evaluated in the same manner as a part of another study, did not contain measureable quantities of this factor.

Increased sciatica on raising the opposite or "well" leg, the crossed straight leg raising (XSLR) sign, is associated with a herniated lumbar disc in 97% of patients. Although XSLR predicts poor response to conservative management, the results of laminectomy are usually good, with 91% of patients returning to work. Myelography is unnecessary for the diagnosis of disc herniation in patients with XSLR. It is possible for patients with this sign to have a normal myelogram, but 90% of them will prove nevertheless to have a herniated disc.

The crystal structure of a novel nucleoside isolated from guanosine/p-methylbenzyl chloride reactions demonstrates linkage between the methylene carbon of the benzyl moiety and carbon-5 of guanosine, and loss of the carbonyl function at carbon-6 of guanosine, to yield 4-(p-methylbenzyl)-5-guanidino-1-beta-D-ribofurasylimidazole. These findings suggest that carbon-5 of guanine in DNA is a potential site of reaction for electrophilic ultimate carcinogens.

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.

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.

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.

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.

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.

Extracts of serum-free conditioned medium from human rhabdomyosarcoma A673 cells contain high molecular weight (HMW) transforming growth factors (TGFs) that can be partially purified by Bio-Gel P-100 and carboxymethyl (CM)-cellulose chromatography (Todaro et al: Proc Natl Acad Sci USA 77:5258, 1980). Reverse-phase high performance liquid chromatography (HPLC) revealed a principal peak of epidermal growth factor (EGF) radioreceptor assay (RRA) activity and anchorage-independent growth (AIG) activity that coeluted with 25-26% acetonitrile. If a trailing shoulder of EGF RRA activity from the CM-C chromatography was included in the material for HPLC analysis, additional active fractions were observed at 21-22% acetonitrile. Importantly, both active regions from HPLC failed to compete in radioimmunoassays under reduced and denatured conditions for human EGF (hEGF), human TGF-alpha (hTGF-alpha), or rat TGF-alpha (rTGF-alpha) and failed to give positive signals in Western blots under conditions in which TGF-alpha was readily detected when using an antisera raised against the 17 C-terminal amino acids of rTGF-alpha. Nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed EGF RRA and AIG activities in gel slices corresponding to Mr 15,000 and 22,000 in the 25-26% acetonitrile eluate and Mr 15,000, 20,000, 27,000, and 48,000 in the 21-22% acetonitrile eluate. The presence of multiple forms of EGF-receptor-binding peptides produced in vitro suggest size heterogeneity and possible immunologic diversity among high molecular weight members of the EGF/TGF-alpha family of growth-promoting polypeptides.