Current Understanding of the Pathophysiology of Osteonecrosis of the Jaw.

PURPOSE OF REVIEW: Osteonecrosis of the jaw (ONJ) is a rare and severe necrotic bone disease reflecting a compromise in the body's osseous healing mechanisms and unique to the craniofacial region. Antiresorptive and antiangiogenic medications have been suggested to be associated with the occurrence of ONJ; yet, the pathophysiology of this disease has not been fully elucidated. This article raises the current theories underlying the pathophysiology of ONJ. RECENT FINDINGS: The proposed mechanisms highlight the unique localization of ONJ. The evidence-based mechanisms of ONJ pathogenesis include disturbed bone remodeling, inflammation or infection, altered immunity, soft tissue toxicity, and angiogenesis inhibition. The role of dental infections and the oral microbiome is central to ONJ, and systemic conditions such as rheumatoid arthritis and diabetes mellitus contribute through their impact on immune resiliency. Current experimental studies on mechanisms of ONJ are summarized. The definitive pathophysiology is as yet unclear. Recent studies are beginning to clarify the relative importance of the proposed mechanisms. A better understanding of osteoimmunology and the relationship of angiogenesis to the development of ONJ is needed along with detailed studies of the impact of drug holidays on the clinical condition of ONJ.

Significance of histologic pattern of carcinoma and sarcoma components on survival outcomes of uterine carcinosarcoma.

BACKGROUND: To examine the effect of the histology of carcinoma and sarcoma components on survival outcome of uterine carcinosarcoma. PATIENTS AND METHODS: A multicenter retrospective study was conducted to examine uterine carcinosarcoma cases that underwent primary surgical staging. Archived slides were examined and histologic patterns were grouped based on carcinoma (low-grade versus high-grade) and sarcoma (homologous versus heterologous) components, correlating to clinico-pathological demographics and outcomes. RESULTS: Among 1192 cases identified, 906 cases were evaluated for histologic patterns (carcinoma/sarcoma) with high-grade/homologous (40.8%) being the most common type followed by high-grade/heterologous (30.9%), low-grade/homologous (18.0%), and low-grade/heterologous (10.3%). On multivariate analysis, high-grade/heterologous (5-year rate, 34.0%, P = 0.024) and high-grade/homologous (45.8%, P = 0.017) but not low-grade/heterologous (50.6%, P = 0.089) were independently associated with decreased progression-free survival (PFS) compared with low-grade/homologous (60.3%). In addition, older age, residual disease at surgery, large tumor, sarcoma dominance, deep myometrial invasion, lymphovascular space invasion, and advanced-stage disease were independently associated with decreased PFS (all, P < 0.01). Both postoperative chemotherapy (5-year rates, 48.6% versus 39.0%, P < 0.001) and radiotherapy (50.1% versus 44.1%, P = 0.007) were significantly associated with improved PFS in univariate analysis. However, on multivariate analysis, only postoperative chemotherapy remained an independent predictor for improved PFS [hazard ratio (HR) 0.34, 95% confidence interval (CI) 0.27-0.43, P < 0.001]. On univariate analysis, significant treatment benefits for PFS were seen with ifosfamide for low-grade carcinoma (82.0% versus 49.8%, P = 0.001), platinum for high-grade carcinoma (46.9% versus 32.4%, P = 0.034) and homologous sarcoma (53.1% versus 38.2%, P = 0.017), and anthracycline for heterologous sarcoma (66.2% versus 39.3%, P = 0.005). Conversely, platinum, taxane, and anthracycline for low-grade carcinoma, and anthracycline for homologous sarcoma had no effect on PFS compared with non-chemotherapy group (all, P > 0.05). On multivariate analysis, ifosfamide for low-grade/homologous (HR 0.21, 95% CI 0.07-0.63, P = 0.005), platinum for high-grade/homologous (HR 0.36, 95% CI 0.22-0.60, P < 0.001), and anthracycline for high-grade/heterologous (HR 0.30, 95% CI 0.14-0.62, P = 0.001) remained independent predictors for improved PFS. Analyses of 1096 metastatic sites showed that carcinoma components tended to spread lymphatically, while sarcoma components tended to spread loco-regionally (P < 0.001). CONCLUSION: Characterization of histologic pattern provides valuable information in the management of uterine carcinosarcoma.

Pelvic squamous cell carcinoma of unknown primary: a case report and review of the literature.

Retroperitoneal squamous cell carcinoma (SCC) of unknown primary is very rare with variable survival rates. Standard optimal therapeutic management is not yet established.

Systemic azithromycin versus amoxicillin/metronidazole as an adjunct in the treatment of periodontitis: a systematic review and meta-analysis.

BACKGROUND: The use of systemic azithromycin (AZT) and amoxicillin/metronidazole (AMX/MTZ) as adjuncts provided additional clinical and microbiological benefits over subgingival instrumentation alone. However, the superiority of one antibiotic regimen over another has not been proven. Therefore, the aim of this systematic review and meta-analyses was to evaluate the clinical efficacy and safety of subgingival instrumentation (SI) in conjunction with the systemic use of AZT or AMX/MTZ for the treatment of periodontitis from current published literature. METHODS: Electronic databases were searched to identify randomized controlled trials (RCTs), controlled clinical trials, prospective and retrospective human studies that compared the adjunctive use of systemic AZT to AMX/MTZ with SI in the treatment of periodontitis. The eligibility criteria were defined based on the participant (who had periodontitis), intervention (SI with adjunctive use of systemic AZT), comparison (SI with adjunctive use of systemic AMX/MTZ), outcomes (primary outcome: changes in probing pocket). The risk of bias was assessed using the Cochrane Collaboration's Risk of Bias tool. Data were analysed using a statistical software program. RESULTS: Five studies with 151 participants with periodontitis were included in the present review. Of these, 74 participants received adjunctive AZT, while the remaining participants received AMX/MTZ as an adjunct to SI. The adjunctive use of AZT and AMX/MTZ had comparable changes in probing pocket depths at 1-3 months with no statistically significant difference (mean difference (MD) 0.01; 95% CI -0.20 to 0.22; P = 0.94). The adjunctive use of AZT had significantly fewer number of residual sites with probing pocket depths of ≥5 mm at 1-3 months compared to the adjunctive use of AMX/MTZ (MD -3.41; 95% CI -4.73 to -2.10; P < 0.0001). The prevalence rates of adverse events among participants who received AZT and AMX/MTZ were 9.80% and 14.8%, respectively. The meta-analysis showed that the difference between the two groups was not statistically significant (risk ratio 0.69; 95% CI 0.28 to 1.72; P = 0.43). CONCLUSIONS: Within the limitation of this review, there was no superiority between AZT and AMX/MTZ in terms of mean changes in probing pocket depths, clinical attachment level, bleeding on probing at 1-3 months. AZT seem to be associated with less sites with residual probing pocket depths of ≥5 mm at 1-3 months and fewer adverse events compared with AMX/MTZ. © 2023 Australian Dental Association.

In vitro analysis of ovarian cancer response to cisplatin, carboplatin, and paclitaxel identifies common pathways that are also associated with overall patient survival.

BACKGROUND: Carboplatin and cisplatin, alone or in combination with paclitaxel, have similar efficacies against ovarian cancer (OVCA) yet exhibit different toxicity profiles. We characterised the common and unique cellular pathways that underlie OVCA response to these drugs and analyse whether they have a role in OVCA survival. METHODS: Ovarian cancer cell lines (n=36) were treated with carboplatin, cisplatin, paclitaxel, or carboplatin-paclitaxel (CPTX). For each cell line, IC(50) levels were quantified and pre-treatment gene expression analyses were performed. Genes demonstrating expression/IC(50) correlations (measured by Pearson; P<0.01) were subjected to biological pathway analysis. An independent OVCA clinico-genomic data set (n=142) was evaluated for clinical features associated with represented pathways. RESULTS: Cell line sensitivity to carboplatin, cisplatin, paclitaxel, and CPTX was associated with the expression of 77, 68, 64, and 25 biological pathways (P<0.01), respectively. We found three common pathways when drug combinations were compared. Expression of one pathway ('Transcription/CREB pathway') was associated with OVCA overall survival. CONCLUSION: The identification of the Transcription/CREB pathway (associated with OVCA cell line platinum sensitivity and overall survival) could improve patient stratification for treatment with current therapies and the rational selection of future OVCA therapy agents targeted to these pathways.

Cyclooxygenase-2 (COX-2) expression in human endometrial carcinoma and precursor lesions and its possible use in cancer chemoprevention and therapy.

In recent years, the design of new antineoplastic agents that can halt the progression of human malignancies with minimal systemic damage has been at the forefront of cancer research, with cyclooxygenase-2 (COX-2) as a major target molecule. With an aim to demonstrate the expression and role of COX-2, the principal putative target of COX-2 inhibitor therapy, in endometrial adenocarcinoma (EACA) and precursor lesions, atypical complex hyperplasia (ACH) and endometrial hyperplasia (EH), an immunohistochemical (IHC) analysis of 22 primary human EACAs and 14 precursor lesions was carried out. Relevant clinicopathological data were tabulated from a random computer-generated sample of 22 primary EACA patients, treated by hysterectomy at our institution. Representative tumor sections including adjacent precursor lesions and normal endometrium (NE) were immunostained with human monoclonal anti-COX-2. Qualitative and semi-quantitative COX-2 IHC staining scores were determined based on the proportion of immunoreactive cells and the intensity of cytoplasmic COX-2 expression. Fisher's exact test and the Wilcoxon Rank Sum test were used for statistical analysis. Mean patient age was 68 years (range 51-93). All 22 EACAs were of endometrioid type, of which ten (45%) were grade I, eight (36%) grade II and four (18%) were grade III. Overall, four out of nine (44%) EHs, four out of five (80%) ACHs, and 18 out of 22 (88%) EACAs were COX-2 positive. The mean COX-2 IHC scores for EH and EACAs were 33 (SD 24.11) and 76 (SD 54.57), respectively (p = 0.022). Strong or moderate COX-2 expression was observed in 17 out of 22 (77%) adenocarcinomas as compared to two out of 14 (14%) of the precursor lesions (EH and ACH). The areas of adenomyosis were COX-2 positive, while myometrial smooth muscle and normal fallopian tube tissues stained negative for COX-2. The demonstration of frequent and strong expression of COX-2 in human EACAs supports a possible role for COX-2 inhibitors. Furthermore, an increasing expression of COX-2 from EH to invasive EACAs suggests potential usefulness of COX-2 inhibition to halt the progression of precursor lesions to invasive endometrial cancers.

Bladder cancer-associated nuclear matrix proteins.

The early diagnosis of bladder cancer is central to the effective treatment of the disease. Presently, there are no methods available to easily and specifically identify the presence of bladder cancer cells. The prevailing method for the detection of bladder cancer is the identification of bladder cancer cells by morphological examination of exfoliated cells or biopsy material by a pathologist. A hallmark of the malignant or transformed phenotype is an abnormal nuclear shape, the presence of multiple nucleoli, and altered patterns of chromatin organization. Nuclear structural alterations are so prevalent in cancer cells that they are commonly used as markers of transformation for many types of cancer. Nuclear shape is determined by the nuclear matrix, the dynamic skeleton of the nucleus. The nuclear matrix is the structural component of the nucleus that determines nuclear morphology, organizes the DNA in a three-dimensional fashion that is tissue specific, and has a central role in the regulation of a number of nuclear processes, including the regulation of DNA replication and gene expression. Previous investigations into prostate and breast cancer have revealed that nuclear matrix protein (NMP) composition undergoes alterations with transformation and that the nuclear matrix can serve as a marker for the malignant phenotype. In this study, we have identified NMPs with which it is possible to differentiate human bladder tumors from normal bladder epithelial cells. We examined the NMP composition of 17 matched tumor and normal samples from patients undergoing surgery for bladder cancer. We have identified six proteins present in all tumor samples that are not present in the corresponding normal samples and three proteins that are unique to the normal bladder tissues in comparison with the tumor samples. Five of the six bladder cancer-associated proteins were also identified in three human bladder cancer cells lines examined (253j, UMUC-2, and T24). Therefore, we have demonstrated that nuclear matrix composition is able to differentiate bladder cancer from normal bladder tissue and may provide useful tools for early detection and recurrence of the disease. Importantly, these markers may provide valuable tools for cytopathological screening for bladder carcinoma.

Benzamide-DNA interactions: deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct.

The interaction of benzamide with the isolated components of calf thymus poly(ADP-ribose) polymerase and with liver nuclei has been investigated. A benzamide-agarose affinity gel matrix was prepared by coupling o-aminobenzoic acid with Affi-Gel 10, followed by amidation. The benzamide-agarose matrix bound the DNA that is coenzymic with poly(ADP-ribose) polymerase; the matrix, however, did not bind the purified poly(ADP-ribose) polymerase protein. A highly radioactive derivative of benzamide, the 125I-labelled adduct of o-aminobenzamide and the Bolton-Hunter reagent, was prepared and its binding to liver nuclear DNA, calf thymus DNA and specific coenzymic DNA of poly(ADP-ribose) polymerase was compared. The binding of labelled benzamide to coenzymic DNA was several-fold higher than its binding to unfractionated calf thymus DNA. A DNA-related enzyme inhibitory site of benzamide was demonstrated in a reconstructed poly(ADP-ribose) polymerase system, made up from purified enzyme protein and varying concentrations of a synthetic octadeoxynucleotide that serves as coenzyme. As a model for benzamide binding to DNA, a crystalline complex of 9-ethyladenine and benzamide was prepared and its X-ray crystallographic structure was determined; this indicated a specific hydrogen bond between an amide hydrogen atom and N-3 of adenine. The benzamide also formed a hydrogen bond to another benzamide molecule. The aromatic ring of benzamide does not intercalate between ethyladenine molecules, but lies nearly perpendicular to the planes of stacking ethyladenine molecules in a manner reminiscent of the binding of ethidium bromide to polynucleotides. Thus we have identified DNA as a site of binding of benzamide; this binding is critically dependent on the nature of the DNA and is high for coenzymic DNA that is isolated with the purified enzyme as a tightly associated species. A possible model for such binding has been suggested from the structural analysis of a benzamide-ethyladenine complex.

Mechanisms of poly(ADP-ribose) polymerase catalysis; mono-ADP-ribosylation of poly(ADP-ribose) polymerase at nanomolar concentrations of NAD.

Calf thymus and rat liver poly(ADP-ribose) polymerase enzymes, and the polymerase present in extracts of rat liver nuclei synthesize unstable mono-ADP-ribose protein adducts at 100 nM or lower NAD concentrations. The isolated enzyme-mono-ADP-ribose adduct hydrolyses to ADP-ribose and enzyme protein at pH values slightly above 7.0 indicating a continuous release of ADP-ribose from NAD through this enzyme-bound intermediate under physiological conditions. NH2OH at pH 7.0 hydrolyses the mono-ADP-ribose enzyme adduct. Desamino NAD and some other homologs at nanomolar concentrations act as 'forward' activators of the initiating mono-ADP-ribosylation reaction. These NAD analogs at micromolar concentrations do not affect polymer formation that takes place at micromolar NAD concentrations. Benzamides at nanomolar concentrations also activate mono-ADP-ribosylation of the enzyme, but at higher concentrations inhibit elongation at micromolar NAD as substrate. In nuclei, the enzyme molecule extensively auto-ADP-ribosylates itself, whereas histones are trans-ADP-ribosylated to a much lower extent. The unstable mono-ADP-ribose enzyme adduct represents an initiator intermediate in poly ADP-ribosylation.

Catalytic activities of synthetic octadeoxyribonucleotides as coenzymes of poly(ADP-ribose) polymerase and the identification of a new enzyme inhibitory site.

The catalytic activity of highly purified poly(ADP-ribose) polymerase was determined at constant NAD+ concentration and varying concentrations of sDNA or synthetic octadeoxyribonucleotides of differing composition. The coenzymic activities of deoxyribonucleotides were compared in two ways: graphic presentation of the activation of poly(ADP-ribose) polymerase in the presence of a large concentration range of deoxyribonucleotides and by calculating kD values for the deoxyribonucleotides. As determined by method i, auto-mono-ADP-ribosylation of the enzyme protein at 25 nM NAD+ was maximally activated at 1:1 octamer/enzyme molar ratios by the octadeoxyribonucleotide derived from the regulatory region of SV40 DNA (duplex C). At a 0.4:1 sDNA/enzyme ratio, sDNA was the most active coenzyme for mono-ADP-ribosylation. At 200 microM NAD+, resulting in polymer synthesis and with histones as secondary polymer acceptors, duplex C was the most active coenzyme, and the octamer containing the steroid hormone receptor binding consensus sequence of DNA was a close second, whereas sDNA exhibited an anomalous biphasic kinetics. sDNA was effective on mono-ADP-ribosylation at a concentration 150-200 -times lower than on polymer formation. When comparison of deoxyribonucleotides was based on method ii (kD values), by far the most efficiently binding coenzyme for both mono and polymer synthesis was sDNA, followed by duplex C, with (dA-dT)8 exhibiting the weakest binding. The synthetic molecule 6-amino-1,2-benzopyrone (6-aminocoumarin) competitively inhibited the coenzymic function of synthetic octadeoxyribonucleotides at constant concentration of NAD+, identifying a new inhibitory site of poly(ADP-ribose) polymerase.

Destabilization of Zn2+ coordination in ADP-ribose transferase (polymerizing) by 6-nitroso-1,2-benzopyrone coincidental with inactivation of the polymerase but not the DNA binding function.

6-Nitroso-1,2-benzopyrone, an oxidation product of 6-amino-1,2-benzopyrone, binds to the DNA-recognizing domain of the ADP-ribose transferase protein and preferentially destabilizes Zn2+ from one of the two zinc finger polypeptide complexes present in the intact enzyme, as determined by the loss of 50% of 65Zn2+ from the 65Zn(2+)-isolated protein molecule, coincidental with the loss of 99% of enzymatic activity. The 50% zinc-deficient enzyme still binds to a DNA template, consisting of a 17-mer DNA primer annealed to M13 positive strand, resulting in the blocking of DNA synthesis by the Klenow fragment of Pol I. Auto-poly-ADP-ribosylated ADP-ribose transferase, which is the probable physiological state of this protein in intact cells, does not bind to primer-template DNA and does not block DNA synthesis by the Klenow fragment. On the basis of this in vitro model it is proposed that molecules which inhibit or inactivate ADP-ribose transferase in intact cells can induce significant alteration in DNA structure and replication.

Molecular interactions between poly(ADP-ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding.

The molecular interactions of poly(ADP-ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the 'global' Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP-ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the 'global' Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I-Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S-transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the 'C clamp' model derived from X-ray crystallography.

Reversion of malignant phenotype by 5-iodo-6-amino-1,2-benzopyrone a non-covalently binding ligand of poly(ADP-ribose) polymerase.

A non-covalently binding inhibitory ligand of poly(ADP-ribose) polymerase, 5-iodo-6-amino-1,2-benzopyrone, when incubated at 5-600 microM external concentration with an E-ras-transformed tumorigenic cell line or with human prostatic carcinoma cells for 40 to 60 days converts both cancer cells to a non-tumorigenic phenotype that is characterized by drastic changes in cell morphology, absence of tumorigenicity in nude mice, and a high rate of aerobic glycolysis.

Potential chemotherapeutic activity of 4-iodo-3-nitrobenzamide. Metabolic reduction to the 3-nitroso derivative and induction of cell death in tumor cells in culture.

A C-nitroso prodrug, 4-iodo-3-nitrobenzamide, was synthesized, and its action on a variety of tumor cells of human and animal origin tested. This prodrug was reduced transiently by tumor cells to 4-iodo-3-nitrosobenzamide at a very low rate, which was, however, sufficient to kill tumor cells. The final reduction product was 4-iodo-3-aminobenzamide, and no intermediates accumulated. No toxicity could be observed in hamsters even at 200 mg/kg, given i.p. daily for 7 days. The chemical reactivity of both 4-iodo-3-nitrosobenzamide and its noniodinated homolog with reduced ascorbate yielded the hydroxylamines. With glutathione, 4-iodo-3-aminobenzamide was formed, suggesting glutathione sulfinic acid formation. Confirming earlier studies, 4-iodo-3-nitrosobenzamide inactivated poly(ADP-ribose) polymerase by zinc ejection from the first zinc finger of this nuclear protein. The iodinated nitroso compound was more effective than its iodine-free analog. Selective tumoricidal action appeared to correlate with the reduction of the nitro group to nitroso in tumor cells, and with the previously described subsequent induction of tumor apoptosis by the C-nitroso intermediate. These processes were accelerated by buthionine sulfoximine, which diminishes cellular GSH.

Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH2BP).

Bovine aortic endothelial cells were converted to a highly tumorigenic cell line by transfection with Ha-ras and stimulation with thrombin. Sustained pretreatment with a non-cytotoxic concentration (600 mu M) of 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a lipophilic ligand of poly(ADP-ribose) polymerase, abrogated in vivo tumorigenicity, of 10(5) cells per inoculum an effect which developed progressively during 2 to 6 weeks of drug treatment. The initial action of the drug was cytostasis, consisting of an arrest in prophase, extreme cell enlargement consistent with cytoplasmic hypertrophy, as seen by EM, and dramatic morphologic changes. Although neither DNA, RNA or protein syntheses are directly affected by INH2BP, apparently newly synthesized cellular DNA is degraded by endonucleases, which are upregulated by the inhibition of their poly-ADP-ribosylation. The drug treated cells exhibited greatly increased respiration and aerobic glycolysis, due to an augmentation of,glycolytic and respiratory enzymes in enlarged cells. These responses to the drug were reversible in cell cultures following drug removal, within 5-10 days drug exposure but the progressive loss of tumorigenicity in nude mice that developed after 3-6 weeks of drug exposure of cells, prior to inoculation to nude mice, was not reversible in vivo. Drug treatment produced a sustained 70-80% inhibition of pADPRT in intact cells at 600 mu M extracellular concentration of INH2BP. The prerequisite for the abrogation of tumorigenicity was the maintenance of pADPRT inhibition. The arrest of cell multiplication and a large decrease of Topo I, especially of Topo II and MAP kinase activities occurred without loss of enzyme protein as assayed in cell extracts of drug-treated cells. However INH2BP had no direct effect on these enzymes. Drug treatment down-regulated DNA-methyltransferase, PKC, ODC proteins, diminished cyclin A protein, but the hypophosphorylated form of Rb protein was significantly augmented. None of the enzymatic components of signal pathways so far studied, were directly affected by INH2BP. The inhibition of pADPRT by INH2BP coincided with an induction or activation of alkaline phosphatase and leucyl and glutamyl peptidase. The pADPRT content or the expression of pADPRT gene were not influenced by drug treatment, but the expression of ras gene was completely absent in nontumorigenic drug-treated cells, without a loss of ras gene from genomic DNA. Telomerase activity was not directly influenced by INH2BP treatment when assayed in diluted cell extracts, but the addition of homogeneous pADPRT to cell extracts, to approach physiological concentration of this protein in the cell, inhibited telomerase activity by binding of the polymer-free pADPRT to telomer templates. We conclude that inhibition of pADPRT indirectly down-regulates growth stimulatory signal pathways and sustains growth-arrested cells in culture at a pre-apoptotic threshold which explains the absence of tumorigenicity in vivo.

Structural specificity and tumoricidal action of methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME).

Seventeen homologs and analogs of methyl-3,5-diiodo-4(4'-methoxyphenoxy) benzoate (DIME), a hormonally inactive analog of thyroid hormones, have been synthesized and their antitumor activity scored by assaying their antitumorigenic effect in vivo following pretreatment of E-ras 20 cells with the drug. In vivo feeding of DIME in large doses had a similar antitumor effect on human tumor xenogafts in vivo without noticeable toxicity of DIME. Inhibition of clonogenicity with MDA-MB-231 cells by DIME yielded I-50 values similar to those found in tests measuring cell growth inhibition (median I-50 less than 1.0 mu M) The apparent ultimate signal for cytocidal action of DIME is the development of dose-dependent double strand cuts of DNA. All human tumor cells so far tested, with the exception of A-549 cells (lung cancer), show high sensitivity to DIME (I-50 1.0 mu M or below). The partially refractory behavior of A-549 cells to DIME is due to the presence of an esterase that cleaves the methyl ester group of DIME. Other tumor cells have negligible esterase activity, while such activity in homogenates of normal mouse tissues is high. Cells in culture take up DIME and the magnitude of uptake parallels drug sensitivity to DIME. Extrapolation from the correlations between drug efficacy, drug uptake, esterase activity and the absence of significant in vivo toxicity of DIME point to uptake of DIME by cells in culture but not by normal cells operating in intact organs in vivo. In contrast, tumor cells in vivo take up DIME and succumb to its cytocidal action just like cells in culture.

Expression of insulin-like growth factor-1 receptor in human colorectal cancer.

The activation of the insulinlike growth factor 1/IGF-1 receptor system (IGF1/IGF1-R) has recently emerged as critical event in transformation and tumorigenicity of several murine and human tumors. Expression of IGF1 and of IGF1-R has been demonstrated in normal and neoplastic intestinal cell lines of rats and humans. However, the modulation of IGF1-R expression during the progression from normal colonic mucosa to adenoma, to carcinoma, and to metastasis, has not been evaluated. In this retrospective study, we investigated the expression of IGF1-R in 12 colonic adenomas (AD), 36 primary colorectal adenocarcinomas (CA), and in 27 corresponding metastases (MT). Normal colonic mucosa (N) was adjacent to the CA in 34 cases. Formalin-fixed, paraffin-embedded tissues of each case were immunostained using the avidin-biotin-peroxidase method. We used an anti-IGF1-R rabbit polyclonal antibody (Santa Cruz Biotechnology, CA; dilution 1:100). Positive staining was quantitated by CAS-200. Moderate to strong cytoplasmic immunostaining was observed in 34 of 36 CA (96%), and in 25 of 27 MT (93%). In all of the positive MTs, the intensity of the staining was always strong. In 10 of 12 ADs (83%), only a faint cytoplasmic stain was identified. Normal mucosa when present was negative. Strong IGF1-R positivity correlated with higher grade and higher-stage tumors (P < .01). These data suggest a role of IGF1-R expression during the progression of colorectal adenoma to carcinoma. An increased number of IGF1-R receptors may favor the metastasis of colorectal cancer.

The Mainz Classification of Renal Cell Tumors.

BACKGROUND: Tumors arising from the renal tubular epithelium have variable characteristics and have been subject to a variety of histologic classifications. METHODS: The authors describe the distinct clinical, pathologic, phenotypic, and genotypic features of different types of renal tumors. RESULTS: The Mainz classification is now widely accepted because characteristic genetic alterations have been demonstrated in each tumor type. CONCLUSIONS: The increasing emphasis on utilizing genetic characteristics of specific tumors is reflected by the more widespread use of the Mainz classification for renal cell tumors.

Value of multilevel sectioning for improved detection of micrometastases in sentinel lymph nodes in invasive squamous cell carcinoma of the vulva.

UNLABELLED: Clinical usefulness of sentinel lymph node (SLN) biopsy has been demonstrated in the management of early vulvar cancer. However, what constitutes a negative SLN has not been well defined. Furthermore, to what extent the SLNs should be sectioned for the greatest likelihood of detection of micrometastases and whether multilevel sectioning will further increase this detection rate in this setting have not been well studied. We analyzed 280 groin lymph nodes (SLNs=45, non-sentinel [NSLNs]=235) in 14 patients with invasive squamous cell carcinoma (ISCC) of the vulva treated with vulvectomy and inguinal SLN and NSLN dissection at the H. Lee Moffitt Cancer Center (HLMCC) between 1996 and 2001. Each SNL was evaluated for micrometastases by H&E and pancytokeratin AE1/3 (CKAE1/3) immunohistochemical staining. All negative SNLs (N=40) were sectioned times 3 (x3) at 50-micron intervals and independently reviewed by two pathologists in order to assess the utility of this inexpensive and logical approach to identifying additional micrometastases. Also, the Wilcoxon Rank Sum Test was used to determine if there was an association between tumor size, depth of invasion and SNL status. The patient age ranged from 35 to 81 years (mean 59 yrs); size of invasive tumor from 1.0 to 7.0 cm (mean 3.4 cm); depth of invasion from 3 to 25 mm (mean 10.8 mm). Of 45 SLNs examined from 14 patients, 11% (5/45) SNLs were positive for micrometastases on initial H&E and/or CKAE1/3 stains. Eighty-nine per cent (40/45) SNLs were negative in the remaining 9 patients. None of the latter 40 SNLs showed micrometastases on additional multilevel sectioning. Instead 3 of 135 NSLNs examined in these 9 patients revealed micrometastases on H&E (skip-micrometastases). Mean tumor size (cm) and depth of invasion (cm) were 4.06 (s.d. 1.89) and 1.20 (s.d. 0.35) for SLN (+) and 3.02 (s.d. 2.12) and 1.01 (s.d. 0.86) for SLN (-) tumor subsets (p values 0.385 and 0.348, respectively). CONCLUSION: Following routine H&E and CK AE1/3 stains, multilevel sectioning does not appear to detect additional micrometastases in sentinel lymph nodes in squamous cell carcinoma of the vulva. Even though mean tumor size and depth of invasion were greater in SNL (+) as compared to SLN (-) tumor subsets in our series, this difference did not reach statistical significance.

Structure elucidation of a 6-methylbenzo[a]pyrene-DNA adduct formed by horseradish peroxidase in vitro and mouse skin in vivo.

Activation of polycyclic aromatic hydrocarbons (PAH) by horseradish peroxidase (HRP) with H2O2 has been studied as a model system for one-electron oxidation. This peroxidase has been used to catalyze binding of 6-[14C]methylbenzo[a]pyrene (BP-6-CH3) to DNA, which was purified, hydrolyzed to deoxyribonucleosides and analyzed by high pressure liquid chromatography (HPLC). The predominant hydrocarbon-DNA adduct observed was identified as BP-6-CH3 bound at the 6-methyl group to the 2-amino group of dG, confirming that activation by HRP occurs by one-electron oxidation. When DNA from mouse skin treated in vivo with [14C]BP-6-CH3 was purified, hydrolyzed and analyzed by HPLC, a profile was observed which was qualitatively similar to that from the peroxidase system. In particular, the identified adduct with the hydrocarbon bound at the 6-methyl group to the 2-amino group of dG was obtained. These results demonstrate that one-electron oxidation is the mechanism of activation by HRP for aromatic hydrocarbons and indicate that the same mechanism may occur in mouse skin, a target tissue for hydrocarbon carcinogenesis.