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Previously, a specific dietary supplement, selected vegetables (SV), was found to be associated with prolonged survival of stage III and IV non-small cell lung cancer (NSCLC) patients. In this study, several anticancer components in SV were measured; the anticancer activity of SV was assessed using a lung tumor model, line 1 in BALB/c mice. SV was also used in conjunction with conventional therapies by stage IIIB and IV NSCLC patients whose survival and clinical responses were evaluated. A daily portion (283 g) of SV was found to contain 63 mg of inositol hexaphosphate, 4.4 mg of daidzein, 2.6 mg of genistein, and 16 mg of coumestrol. Mouse food containing 5% SV (wt/wt) was associated with a 53-74% inhibition of tumor growth rate. Fourteen of the 18 patients who ingested SV daily for 2-46 months were included in the analyses; none showed evidence of toxicity. The first lead case remained tumor free for > 133 months; the second case showed complete regression of multiple brain lesions after using SV and radiotherapy. The median survival time of the remaining 12 patients was 33.5 months, and one-year survival was > 70%. The median survival time of the 16 "intent-to-treat" patients (including ineligible patients) was 20 months, and one-year survival was 55%. The Karnofsky performance status of eligible patients was 55 +/- 13 at entry but improved to 92 +/- 9 after use of SV for five months or longer (p < 0.01). Five patients had stable lesions for 30, 30, 20, 12, and 2 months; two of them, whose primary tumor was resected, used SV alone and demonstrated an objective response of their metastatic tumors. In addition to the two lead cases, eight patients had no new metastases after using SV. Three patients had complete regression of brain metastases after using radiotherapy and SV. In this study, daily ingestion of SV was associated with objective responses, prolonged survival, and attenuation of the normal pattern of progression of stage IIIB and IV NSCLC. A large randomized phase III clinical trial is needed to confirm the results observed in this pilot study.

This phase I/II study evaluates the influence of selected vegetables (SV) that contain known antitumor components on the survival of stage III-IV non-small cell lung cancer (NSCLC) patients. All patients were treated with conventional therapies. SV was added to the daily diet of 5 stage I patients in the toxicity study group (TG) and 6 stage III and IV patients in the treatment group (SVG), but not to the diet of 13 stage III and IV patients in the control group (CG). Age, Karnofsky performance status (KPS), and body mass index of SVG and CG patients were comparable at entry. KPS declined in the CG patients (79 +/- 8 to 55 +/- 11) but improved in the SVG patients (75 +/- 8 to 80 +/- 13) one to three months after entry. Weight change in the CG, SVG, and TG patients was -12 +/- 5%, -2 +/- 2%, and +4 +/- 4%, respectively. The median survival time and mean survival of the CG patients were 4 and 4.8 months, but in the SVG patients they were 15.5 and 15 months (p < 0.01). No clinical signs of toxicity were found in the TG patients in the 24-month study period. Adding SV to the daily diet of NSCLC patients was found to be nontoxic and associated with improved weight maintenance, KPS, and survival of stage III and IV NSCLC patients.

Histones are known to bind anionic phospholipids (PLs). Binding of procoagulant PLs by histones released during cell injury/death may interfere with coagulation and may serve a local regulatory anticoagulant function. Histone H1 prolonged the PT and APTT of normal pooled plasma (NPP). These increased clotting times disappeared when anti-H1 monoclonal antibody (mAb) was added to the incubation. Dilute Russell Viper Venom Time was also prolonged with the addition of histone H1. When H1 was added to plasma from a patient with the antiphospholipid syndrome (APL plasma), there was a further prolongation of the abnormal APL clotting time which was partially corrected by anti-H1 mAb. Platelet neutralization times were increased with added H1 and were further increased using APL plasma. when disrupted endothelial cells were incubated with plasma with and without anti-H1 antibodies, the addition of anti-H1 antibodies decreased clotting times. These data support the theory that histones released during cell injury may have a regulatory anticoagulant role in clot formation and the anti-H1 effect of some APL plasmas may inhibit this, thereby contributing to thrombosis seen in APL patients.

The levels of histone-reactive IgA antibodies in the sera of adult patients with IgA mesangial glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis and idiopathic nephrotic syndrome (minimal change disease+segmental glomerulosclerosis+IgM nephropathy) were evaluated by an enzyme-linked immunosorbent assay. Increased levels of IgA antibodies to all five major histones (H1, H2A, H2B, H3, H4) were found in all four disease groups when compared to normal controls. These histone-reactive IgA antibodies were restricted to the IgA1 subclass and their levels did not correlate with the levels of total serum IgA, nor with serum creatinine, creatinine clearance, and 24-hour proteinuria. Increasing ionic strength resulted in only partial inhibition of the binding to histones and, in individual patients, levels of reactivity with individual histones were usually correlated. This study shows that elevated levels of IgA antibodies reactive with self antigens are present in primary glomerulonephritis and extends previous observations indicating that anomalies of the IgA system occur in various forms of primary glomerulonephritis and are not limited to IgA nephropathy.

Objective. To characterize the binding properties and variable-region sequences of LG4-1, a monoclonal antibody from an autoimmune MRL/Mp-lpr/lpr mouse that reacts specifically with nucleosome core particles and represents a new antinuclear antibody specificity. Methods. The reactivity of the antibody against various nuclear substrates was determined using an enzymatic immunoassay, and the variable-region genes were sequenced from messenger RNA, using the dideoxy chain termination method. Results. LG4-1 was found to react with nucleosome core particles but not with individual histones and DNA, or with various histone–histone and histone–DNA complexes. It was demonstrated that this antibody is encoded by a combination of variable-region genes and gene segments that have undergone few somatic mutations. Conclusion. The nucleosome core particle expresses a unique conformational autoepitope(s) resulting from the ordered association of histones and DNA.

Fifty-two patients with ulcerative colitis and colorectal cancer undergoing colectomy at the Mount Sinai Hospital between 1973 and 1988 were studied retrospectively to determine the correlation of age, sex, duration of colitis, tumor location, number of cancers, tumor differentiation, colloid content, presence of signet ring cells, Dukes' classification, and DNA ploidy with survival. The mean age was 45 years, with a mean duration of colitis of 21 years. Five patients (10%) had Dukes' A lesions, 17 (33%) had Dukes' B lesions, 17 (33%) had Dukes' C lesions, and 13 (25%) had distant metastases. Thirty patients (58%) had well- or moderately differentiated tumors, whereas tumors were poorly differentiated in 22 (42%). Twenty-eight patients (54%) had colloid tumors, and, in 14 (27%), signet ring cells were present. Thirty-one patients (60%) had nondiploid tumors. Actuarial analysis revealed that the 5-year survival rate was significantly worse for patients with nondiploid tumors (76% versus 32%). When stratified by stage, only patients with Dukes' C lesions showed a significant difference in survival for diploid versus nondiploid tumors. Multivariate analysis showed that the Dukes' classification was the best prognostic indicator, followed by tumor differentiation and DNA ploidy. Tumor location, colloid content, number of cancers, duration of disease, age, and sex did not correlate with the prognosis.

Lymphomatoid papulosis (LyP) is rarely seen in children. We report a case of LyP in a 4-year-old boy in whom immunopathologic studies demonstrated T cell antigen deletions. In contrast to all but two previous reports, a T suppressor (CD-8) phenotype was predominant. Southern blot analysis of DNA isolated from a typical skin lesion indicated a clonal rearrangement of the T cell receptor beta gene. Because of a 10% frequency of malignant lymphomas in patients with LyP, long-term observation is crucial, especially in children. We recommend routine clonal rearrangement studies for aid in diagnosis and follow-up, and as possible prognostic indicators in children with this condition.

In this report, we tested two commercial preparations of DNase I for their effect on histone H1 and its recognition by a monoclonal anti-H1 antibody. Our results indicate that digestion with the DNase I preparation of a lesser grade results in the complete proteolysis and loss of antigenicity of histone H1 even though this preparation is adversited as containing less than 0.01% chymotrypsin and less than 0.005% protease

Asbestos fibers are widespread environmental carcinogens whose mutagenicity is now established. Nonetheless, the molecular nature of these mutations and the mechanisms by which they accelerate carcinogenesis remain poorly understood. We have assessed the ability of asbestos fibers to promote homologous recombination, a potent mechanism for generating intrachromosomal rearrangements, such as deletions, and mitotic recombination. For this, we have developed a new assay which determines the extent to which a marker gene present in DNA introduced by asbestos can recombine with homologous genes residing in a transfected cell. We have demonstrated that Calidria chrysotile fibers are mutagenic and are able to mediate transfection of molecularly marked mutant lacI genes in a manner that results in their preferential recombination with homologous wild-type genes in the transfected cell. Asbestos induced recombination events may play a significant role in asbestos mutagenesis and carcinogenesis, and promotion of recombination may underlie the well-recognized synergy of asbestos with other carcinogens.

The histone H2A-H2B dimer is a component of nucleosomes in chromatin and a frequent target of autoantibodies in spontaneous and drug-induced lupus. We obtained a panel of several lgG mAbs reacting with H2A-H2B or DNA from MRL mice which develop a spontaneous lupus-like syndrome. Several of these antibodies do not react with individual histones, but bind strongly to the H2A-H2B dimer and some bind even more strongly to the H2A-H2B-DNA complex. Moreover, these antibodies not only bind to H2A-H2B dimers in the absence of DNA, but also exhibit significant binding to DNA in the absence of histones, indicating an overlap between the anti-histone and anti-DNA specificities. The analysis of the variable region gene sequences of these antibodies shows a recurrent usage of similar VH genes, suggesting a dominant role for the heavy chain in determining binding specificity. The heavy chain third complementarity determining regions of these antibodies are also remarkable for their frequency of D-D fusions and of D segments read in unusual reading frames and for many arginine residues that may contribute to DNA binding. In addition, several antibodies obtained from an individual mouse are clonally related and some differ through somatic mutations, indicating that autoreactive clones are positively selected by nuclear antigens.

This study was performed to determine the correlation of tumor ras and c-myc oncogene expression with clinical and prognostic variables in patients prone to develop colorectal cancer. One hundred eighteen patients with colorectal cancer were studied; mean age was 40 years. Fifty-three were young patients (age 40 or less), 49 had ulcerative colitis, and 16 had multiple polyposis coli. Immunoperoxidase stains of paraffin-embedded cancer sections were performed for the c-myc and ras proteins. ras staining was found to correlate with Dukes stage and prognosis. Patients with tumors negative for ras protein stain had an actuarial five-year survival of 61 percent versus 44 percent for those tumors with a positive stain (P less than 0.05). This correlation was not seen with the c-myc stain. Positive ras oncogene stain appears to be a useful indicator of advanced stage and poor prognosis in colorectal cancer occurring in cancer-prone patients.

Sixteen patients with polyposis coli and cancer were studied retrospectively to determine the incidence of DNA ploidy abnormalities in the tumors and synchronous polyps. Six patients (37 percent) had nondiploid tumors. Nondiploid tumors were more likely to be advanced and had a significantly worse prognosis (17 percent vs. 76 percent 5-year survival; P less than 0.01). Only 4 of 20 polyps studied were nondiploid. There was no association between tumor and polyp ploidy. All nondiploid polyps were found in patients with synchronous diploid cancers. Patients with nondiploid polyps were more likely to be older and have more advanced tumors than those with diploid polyps. DNA ploidy abnormalities seem to occur with the same frequency in polyposis coli as in the nonpolyposis population, and tumor ploidy correlates with prognosis.

We investigated the levels of anti-histone antibodies in the sera of 7 patients with subacute sensory neuropathy. IgG antibodies to histones H1 and H3 were significantly elevated in 4 of these patients. The anti-H1 antibodies reacted mainly with determinants located in the central globular and the carboxy-terminal domain of the H1 molecule. We also observed reactivity of these sera with histone H1 zero, a variant found in terminally-differentiated cells such as neurons. This study suggests a potential for histones to serve as autoantigens in humorally-mediated paraneoplastic diseases.

The binding of antinuclear antibody-positive juvenile arthritis (JA) sera to bovine thymus histones H1, H2A, H2B, H3, and H4 was studied by an enzyme-linked immunosorbent assay. Seventy-five percent of the JA patients tested positive for at least 1 antibody specificity. Antihistone antibodies were predominantly IgM, while IgG antibodies were less common and were restricted to histones H1 or H3. In the group of patients with JA of pauciarticular onset, antihistone antibodies were significantly more elevated in patients with past or present uveitis than in patients without a history of uveitis. Anti-H1 antibodies in JA patients were found to react mostly with determinants located in the carboxyl-terminal domain of the H1 molecule. Sera were also reactive with human histone H1(0) or chicken histone H5, which are H1 variants found only in nondividing cells.

Several studies have shown that the presence of DNA content abnormalities, measured by flow cytometry, may correlate with a poor prognosis in a variety of cancers. The predictive value of DNA content in patients with small rectal cancers has not been well determined. Thirty-nine patients with primary rectal adenocarcinoma smaller than 3 cm were studied in a comparison of DNA content with established prognostic variables. The following covariates were evaluated for their prognostic value: sex, age, tumor size, location, distal margin, Dukes' classification, tumor differentiation, and DNA content. DNA content was assessed by flow cytometric analysis, and each tumor was categorized as diploid or nondiploid. Of the parameters studied, Dukes' classification and tumor DNA content were found to be independent prognostic indicators. Determination of DNA content seems to provide additional useful prognostic information in patients with small rectal tumors.

The estrogen receptor (E2R) from rabbit uterus has an enhanced affinity for BrdUrd-substituted DNA compared to unsubstituted DNA. Increasing levels of BrdUrd substitution (from 0 to 100%) in a DNA sample are associated with increasingly tighter binding of E2R to that DNA as measured by equilibrium competition experiments and by rates of dissociation (or receptor transfer experiments). Although the rates of dissociation of E2R-DNA complexes vary greatly, depending on the BrdUrd-substitution level in the DNA, no differences were detected between the rates of association of E2R with unsubstituted and fully BrdUrd-substituted DNA. The E2R-DNA complex dissociates more rapidly in 150 mM KCl than in 50 mM KCl; but, at both ionic strengths, BrdUrd substitution in the DNA confers enhanced stability on the complex. The demonstration that a specific mammalian regulatory protein has an enhanced affinity for BrdUrd-substituted DNA further strengthens the possibility that BrdUrd modulates gene expression through an altered binding of regulatory proteins.

Although the interplay between steroid hormone receptors and the genome has been extensively studied [l-6] , it is not yet known how the receptor protein interacts with double-stranded helical DNA. To examine the specificity and the dynamics of DNA-estrogen receptor (ER) interactions, we have initiated a study to determine how specific changes in the DNA will affect receptor binding. We have found that ER binds preferentially to AT-rich DNA [7] and also to DNA substituted with bromodeoxyuridine (BrdUrd) [8] . Numerous studies, in a wide spectrum of cell systems, have examined various changes in gene expression which follow BrdUrd-substitution in the DNA [9,10] . Relatively few experiments, however, have focused on the possible mechanism(s) by which this defined replacement (of the S-methyl group in thymine by a bromide atom) might cause subtle electronic or steric effects in DNA which, in turn, might alter gene expression in the eukaryotic cell [g-12]. One such mechanism involves altered binding of regulatory proteins to BrdUrd-substituted DNA [g-12]. One approach to the question of how the bromine atom causes its particular effects, is to ask if other halogen atoms in the same position cause the same effects [ 121. Therefore, we have incorporated 5-chloro-, 5-bromoand Siodo-deoxyuridine into DNA in equivalent molar amounts and determined the effect of these substitutions on the ER

Medicine, New York, New York 10035 We have used equilibrium dialysis and other tech- niques to examine the actinomycin D binding sites on unsubstituted and halodeoxyuridine-substituted DNAs. Some of the actinomycin D binding sites present on halodeoxyuridine-substituted DNAs have an enhanced affinity for actinomycin D compared to the standard binding sites present on unsubstituted DNA. Kinetic studies indicate that actinomycin D dissociates from these high affinity sites at a slower rate than it does from the standard sites on unsubstituted DNA. Increas- ing levels of bromodeoxyuridine substitution are asso- ciated with increased binding of actinomycin D to the substituted DNA. Substitution with iododeoxyuridine causes a greater increase in actinomycin D binding than do equivalent levels of substitution with chloro- deoxyuridine or bromodeoxyuridine. Some halodeox- yuridine-substituted DNAs appear to have additional binding sites for actinomycin D which are not available on unsubstituted DNA. The finding, that poly(dA- 5BrdU).poly(dA-5BrdU) as well as poly(dA-dT)

Several different in vitro binding assays show that the estrogen receptor from rabbit uterus interacts selectively with purified histones from calf thymus. The estrogen receptor binds strongly to histones H2B and H2A, moderately to histones H3 and H4, and poorly to histone H1. In the presence of histones H2B or H2A, the position at which the estrogen receptor focuses in an isoelectric gradient is shifted to a more basic zone. Kinetic experiments show that, if histone H2B is bound to a DNA, the estrogen receptor dissociates more slowly from that DNA. The portion of the estrogen receptor molecule required for binding to histone H2B is relatively stable to tryptic digestion; in contrast, the portion of the receptor molecule responsible for DNA binding is promptly lost during limited tryptic digestion. These in vitro findings suggest that the mechanism by which the estrogen receptor selectively alters gene expression may involve specific contacts with histone molecules.

Phosphorylation of H1 histones by cyclic AMP-dependent protein kinase may be an important transcriptional control mechanism. We have used affinity chromatography to examine the effect of phosphorylation by this enzyme on the DNA-binding properties of calf thymus H1 histones and two highly basic H1 homologues from condensed and transcriptionally silent nuclei: duck erythrocyte H5 and Strongylocentrotus purpuratus sperm H1. Without in vitro phosphorylation, all three histones were eluted from native DNA-Sephadex G-25 columns at salt concentrations which closely resembled those required to extract these histones from nuclei or chromatin. When a small portion of radioactively phosphorylated histone was chromatographed with untreated carrier histone, the phosphorylated species was consistently eluted from the DNA column at slightly lower salt concentrations than the main histone peak. Rechromatography experiments showed that in vitro phosphorylation of H1 can shift its elution position to lower salt concentrations.

It has been postulated that polymerized human serum albumin may play a role in the infection of hepatocytes by hepatitis B virus, because both the envelope of hepatitis B virus (HBsAg) and hepatocytes exhibit binding activity for human serum albumin after cross-linking by glutaraldehyde. Since glutaraldehyde-dependent cross-linking of albumin molecules is not likely to occur in vivo, we considered the possibility that albumin may be polymerized by the action of transglutaminase enzymes present in plasma as activated factor XIII or released into plasma from tissues. Guinea pig liver transglutaminase covalently cross-linked human serum albumin molecules into dimers, trimers and polymers up to hexamers as shown by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. HBsAg particles bound transglutaminase-cross-linked as well as glutaraldehyde-cross-linked human serum albumin as demonstrated by radioimmunoassay and immunoelectron microscopy. The binding was blocked by preincubation of HBsAg with transglutaminase- or glutaraldehyde-cross-linked human serum albumin, anti-HBs or monoclonal anti-pre-S2, but not by polymerized bovine or rat serum albumin or by monomeric human serum albumin. These data indicate that HBsAg particles contain specific binding sites for transglutaminase-cross-linked human serum albumin, but it remains to be determined whether the albumin polymers play a role in the attachment of hepatitis B virus to hepatocytes.

Hybridomas producing anti-histone monoclonal antibodies were generated from a 2-month old MRL lpr/lpr mouse. Five IgM antibodies showed binding to histone H3 and three of these IgM antibodies also bound histone H1. Two IgG2a antibodies (MRA3 and MRA12) were specific for histone H1 and their binding was further characterized. Both reacted strongly with mouse and calf thymus histones H1, but showed limited (if any) binding to H1 from non-mammalian species such as duck, trout, or sea urchin sperm. Histone H1 is composed of three domains: N (N-terminal), G (globular) and C (C-terminal). The binding of MRA3 and MRA12 antibodies to the N, G and C domains of the histone H1 molecule was also investigated, using purified H1 fragments. Significant binding was observed only with the GC fragment but not with isolated NG, G or C fragments. Moreover, the integrity of most, if not all, of the G domain (residues 33-122) was necessary for antibody binding, since cleavage of the H1 molecule either at residue 72 or 106 abolished the binding to MRA3 and MRA12. Taken together, these results could indicate that MRA3 and MRA12 antibodies recognize a conformational determinant of the H1 molecule.

A technique for performing mucosal proctectomy in patients with ulcerative colitis using ultrasonic fragmentation is described. Twenty-eight patients undergoing colectomy and ileoanal anastomosis were studied. Removal of the mucosal layer of the distal rectum was performed using a titanium probe vibrating at 23 kHZ with an amplitude of 300 microns. This method produces complete mucosal destruction and the resulting debris and irrigating fluid is removed through the hollow central portion of the probe. Healing of the ileoanal anastomosis does not appear to be adversely affected by the use of this technique. Because ultrasonic fragmentation is not dependent on the integrity of the submucosal plane, it may be advantageous in those cases in which severe inflammation and submucosal scarring make manual dissection of the rectal mucosa difficult to perform.