Complete sequencing of the p53 gene provides prognostic information in breast cancer patients, particularly in relation to adjuvant systemic therapy and radiotherapy.

The complete coding region of the p53 gene was sequenced from 316 consecutively presented breast cancers, of which 97 were lymph node positive and 206 were node negative. The p53 status was related to prognosis and effect of adjuvant therapy. In all, 69 individual mutations, 29 in node-positive tumours, were demonstrated throughout the whole coding sequence. The mutation sites were partly different for node-positive and node-negative patients. p53 mutations in the evolutionary conserved regions II and V were associated with significantly worse prognosis. Adjuvant systemic therapy, especially with tamoxifen, along with radiotherapy seemed to be of less value to p53 mutation- and lymph node-positive tumours.

The orphan receptor GPR55 is a novel cannabinoid receptor.

BACKGROUND: The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes. EXPERIMENTAL APPROACH: Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways. KEY RESULTS: We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1. CONCLUSIONS: These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

The p53 gene in breast cancer: prognostic value of complementary DNA sequencing versus immunohistochemistry.

BACKGROUND: Mutations in the p53 tumor suppressor gene (also known as TP53) have been detected in a wide variety of human cancers. In breast cancer, the presence of p53 gene alterations has been associated with worse prognosis. PURPOSE: We compared a complementary DNA (cDNA)-based sequencing method and an immunohistochemical (IHC) method for their abilities to detect p53 mutations in breast cancer specimens. In addition, we determined the prognostic value of information obtained when these two methods were used. METHODS: Specimens from 316 primary breast tumors were evaluated for the presence of mutant p53 protein by use of the mouse monoclonal antibody Pab 1801 (that recognizes both wild-type and mutant forms of p53) and standard IHC methods. In addition, the entire coding region of p53 genes expressed in these tumors was screened for mutations by combining reverse transcription, the polymerase chain reaction, and DNA sequencing. Probabilities for overall survival (OS), breast cancer-corrected survival (BCCS; death from breast cancer is the considered event), and relapse-free survival (RFS) were estimated by use of the Kaplan-Meier method, and survival curves for different patient subgroups were compared by use of the logrank method. All reported P values are from two-sided tests. RESULTS: Sixty-nine (22%) of 316 tumors had p53 gene mutations detected by the cDNA-based sequencing method; only 31 (45%) of these mutations were located in evolutionarily conserved portions of the p53 coding region. Sixty-four tumors (20% of the total) had elevated levels of p53 protein as detected by IHC, suggesting the presence of mutations. Of the sequencing-positive tumors (i.e., p53 mutant), 23 exhibited negative IHC reactions, indicating that IHC failed to detect 33% of the mutations. Furthermore, 19 of the IHC-positive tumors were sequencing negative (i.e., p53 wild-type), suggesting a 30% false-positive frequency with IHC. Four tumors (1.3% of the total) could not be analyzed by the cDNA-based sequencing method, and three tumors (1% of the total) could not be analyzed by IHC. The 5-year estimates for RFS, BCCS, and OS were significantly shorter for patients with p53 sequencing-positive tumors than for patients with sequencing-negative tumors (P = .001, P = .01, and P = .0003, respectively). Patients with IHC-positive tumors showed reduced survival in all three categories when compared with those with IHC-negative tumors, but the differences were not statistically significant. CONCLUSIONS: Use of a cDNA-based sequencing method to determine the status of the p53 gene in primary breast cancers yielded better prognostic information than IHC performed with the Pab 1801 monoclonal antibody.

Prognostic and predictive value of c-erbB-2 overexpression in primary breast cancer, alone and in combination with other prognostic markers.

PURPOSE: To investigate the prognostic and predictive value of c-erbB-2 overexpression in breast cancer in relation to other prognostic markers. PATIENTS AND METHODS: Paraffin-embedded tumors from 315 consecutive primary breast cancer patients were screened for c-erbB-2 protein (p185) overexpression by immunohistochemistry using the monoclonal antibody CB11. RESULTS: c-erbB-2 protein overexpression was detected in 19% of tumors and was associated with shorter 5-year overall survival (OAS) rate compared with c-erbB-2-negative cases in the total patient material (58% and 77%, respectively; P = .004) and in the 96 node-positive patients (31% and 61%, respectively; P = .02), but not in node-negative patients. For 47 node-positive patients treated with adjuvant tamoxifen and radiotherapy, the 5-year OAS was 13% for c-erbB-2 overexpression and 75% for c-erbB-2-negative patients (P = .00004). The frequency of c-erbB-2 overexpression decreased with age at diagnosis. The prognostic value of c-erbB-2 on OAS was independent of age, node status, tumor size, histopathologic grade, hormone receptor status, S phase, p53 status, and adjuvant treatment. c-erbB-2 status added prognostic information to p53-negative and low S-phase cases, but not to p53-positive and high S-phase cases. Correspondingly, these only added information to c-erbB-2-negative cases. CONCLUSION: c-erbB-2 protein overexpression may have a predictive value with regard to adjuvant therapy in node-positive patients, for whom adjuvant tamoxifen with radiotherapy appears insufficient in the presence of c-erbB-2 overexpression. Combination of conventional and newer tumor markers may identify patients with a worse prognosis within groups with a generally favorable prognosis.

p53 Status predicts survival in breast cancer patients treated with or without postoperative radiotherapy: a novel hypothesis based on clinical findings.

PURPOSE AND METHODS: Primary breast cancer tumors without axillary metastases from 206 consecutive patients in a population-based cohort were investigated with regard to the presence of an intact p53 gene using a cDNA-based sequencing method. Clinical follow-up data and outcome of node-negative patients without any adjuvant systemic therapy (n = 168) were related to locoregional radiotherapy and p53 status. RESULTS: Mutations in p53 occurred in 31 node-negative breast cancer patients who did not receive any systemic adjuvant treatment, but were treated with postoperative locoregional radiotherapy or nothing. Node-negative breast cancer patients with p53 mutations had significantly improved relapse-free survival (P = .0007), breast cancer-corrected survival (P = .01), and overall survival (P = .02) rates when treated with locoregional radiotherapy. In node-negative breast cancer patients with wild-type p53, there was no statistically significant difference in outcome between patients who received locoregional radiotherapy and those who did not. Cox proportional hazards models indicate that mutant p53 is associated with worse prognosis independent of response to radiotherapy and that response to radiotherapy is qualitatively different in tumors with p53 mutations compared with those with wild-type p53. CONCLUSION: Our clinical findings define a group of breast cancer patients in whom locoregional radiotherapy improves relapse-free, breast cancer-corrected, and overall survival. The outcome for irradiated node-negative breast cancer patients with p53 alterations indicates that irradiation can induce cell death even in the presence of p53 mutations.

Changes induced in growing rat bone by immobilization and remobilization.

We studied changes in bone mass and histology in growing rats after different relatively short periods of immobilization and during subsequent remobilization. Immobilization-induced loss of bone weight is mainly due to mineral losses as indicated by changes in wet weight, ash weight, and calcium content. 45Ca2+ incorporation was found to be decreased in immobilized bones and showed strong dependence upon the age of the rats. Histological examination showed rapid and extensive trabecular bone loss, and external measurements of bone length and diameter confirmed that a substantial part of the decrease in bone mass was due to actual trabecular bone loss and not the reduction of external bone volume. Two of the methods studied, cast immobilization and reversible neurectomy, allow subsequent remobilization and thus enable recovery of the bone to be studied. Bone ash weights were 12.3 +/- 1.12% and 13.1 +/- 1.82% below the control values in the tibia and the femur, respectively, after three weeks of cast immobilization and 12.0 +/- 1.10% and 9.2 +/- 0.90% below after three weeks of immobilization by reversible neurectomy. The bone mineral mass recovered by 40% (p less than 0.053) in the femur and 67% (p less than 0.027) in the tibia during the three weeks' remobilization following one week of cast immobilization, and 62% (p less than 0.001) in the tibia but only 38% (p less than 0.073) in the femur after three weeks of cast immobilization. Mobility of the extremity was restored after three weeks of immobilization by reversible neurectomy, whereupon about half of the lost bone mass was recovered in both the tibia and the femur during six weeks of reinnervation.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitonin gene-related peptide in rat salivary glands: neuronal localization, depletion upon nerve stimulation, and effects on salivation in relation to substance P.

Calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibres occurred predominantly around blood vessels and large ducts and, to a minor extent, around acini and small ducts in the parotid, sublingual and submaxillary glands of the rat. Double immunostaining showed most of the CGRP-containing nerve fibres to contain substance P. However, the vast majority of substance P-immunoreactive periacinar nerve fibres in the parotid and submandibular glands lacked CGRP. After parasympathetic denervation of the parotid gland by section of the auriculotemporal nerve these periacinar substance P-immunoreactive nerve fibres disappeared almost completely, whereas the number of substance P/CGRP-immunoreactive nerve fibres seemed unchanged. After this operation the total amount of substance P in the parotid gland was reduced by about 90% as judged by radioimmunoassay; in denervation experiments the facial nerve was found to contribute to the residual substance P content. In contrast, the contribution of the auriculotemporal nerve to the CGRP content of the gland was small; the reduction in CGRP after section of the nerve was 20%. The facial nerve and the dorsal root nerves (C3 and C4) contributed to the CGRP content with about 50%. The source of the remaining 30% of the parotid gland CGRP is unknown. It is not the sympathetic nerve: sympathetic denervation resulted in a marked increase in CGRP, regardless of whether the auriculotemporal nerve was intact or not. Upon long-lasting electrical stimulation of the auriculotemporal nerve at a high frequency the parotid gland content of CGRP was gradually reduced, indicating depletion of this peptide in response to nerve stimulation. Intravenous injections of CGRP evoked no salivary flow; however, a release of amylase was revealed. Also, when CGRP was tested on isolated parotid gland lobules amylase was released into the medium. When, in vivo, CGRP was injected in combination with substance P, the substance P-evoked flow of parotid and submaxillary saliva was markedly enhanced. In addition, CGRP enhanced the in vivo secretory response to parasympathomimetics and to vasoactive intestinal peptide. The localization of CGRP-containing nerve fibres suggests that CGRP is involved in the regulation of secretion and blood flow of salivary glands. CGRP may interact positively with acetylcholine and certain nonclassical transmitters, and it may be involved (together with other neuropeptides) in the atropine-resistant parasympathetic secretion occurring in the glands under study.

Lactate dehydrogenase in developing rat oral epithelium.

Freeze-dried sagittal, whole-body sections of 10-day-old rats were incubated for lactic dehydrogenase (LDH) using different media in the presence of the inhibitors urea and fluoropyruvate. Phenazine methosulfate (PMS) and menadione, which are regularly used in current histochemical media and are believed to promote the demonstration of LDH activity, were also added and shown to be insufficient for the demonstration of total LDH activity, and PMS even seemed to have an inhibitory effect on LDH activity in oral epithelium. However, cumulated data from the different incubations show that the oral epithelium of developing rats may contain two different types of LDH, one in the basal cells with possibly aerobic characteristics, and another in the spinosum/granulosum cells with anaerobic characteristics.

Acid, neutral, and alkaline phosphatases in developing rat oral mucosa.

Developing rat oral mucosa contains high activities of acid (AcidPase), neutral (adenosine monophosphatase AMPase), and alkaline (AlkPase) phosphatases. This study is concerned with a detailed analysis of the distribution of these enzymes in freeze-dried sections of oral mucosa. The sections were incubated for AcidPase. AMPase, and AlkPase in the presence of certain inhibiting agents. Four AcidPases, three AMPases, and two AlkPases were identified in the oral mucosa. Six of them were found in the epithelium and three in the connective tissue. Three of the epithelial phosphatases were unique in the sense that they were found only in junction mucosa epithelium. The function of these enzymes may be related to the maintenance of the epithelial cell attachment in an area subjected to stretching, possibly by affecting the periphery of the cells.

Lactate dehydrogenase isozymes in developing rat oral mucosa. A comparative study of LDH biochemistry and histochemistry.

Histochemical lactic acid dehydrogenase (LDH) staining methods seem unable to demonstrate the total LDH activity in tissue sections. An analysis was made of LDH tissue staining methods applied on LDH zymograms. The menadione-mediated LDH staining of tissue sections can not possibly reflect true LDH activity. The addition of cyanide also slightly inhibited LDH activity. The cyanide inhibition was confirmed via LDH assay and found to be competitive in character. It is concluded that cyanide and menadione should be replaced by agents suitable from both a histochemical and a biochemical point of view. Based on the findings of this study the presence of LDH in oral epithelium was analyzed. Evidently LDH of the oral epithelium is basically anaerobic in character and located primarily in spinosum/granulosum layers and only sparsely in the basal layer.

Enzyme histochemistry of developing rat oral mucosa.

The oral mucosa of developing and mature rats was analyzed histochemically for regional enzyme differences. The following enzymes were studied: nonspecific alkaline phosphatase (alkpase), acid phosphatase (acidpase), 5'-nucleotidase (AMPase), adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G-6-pDH). All enzymes were active in the oral mucosa, but regional as well as tissue variations were observed. Epithelium in all regions showed acidpase staining. Oxidoreductases were found in all regions with variations within the epithelium. The epithelium of specific regions stained for alkpase and AMPase, while adjacent epithelium did not. We suggest that the alkpase and AMPase activities are associated with specific functions of the epithelium in these regions.

Tyrosine hydroxylase in mouse pancreatic islet cells, in situ and after syngeneic transplantation to kidney.

Tyrosine hydroxylase (TH) is co-expressed with islet hormones in the fetal mouse pancreas. In the adult animal, the enzyme has been considered as a marker of ageing beta-cells. By immunohistochemical staining, we analyzed the expression of TH-like immunoreactivity (TH-LI), insulin-LI (INS-LI) and somatostatin-LI (SOM-LI) in adult mouse islets, in situ and after isolation and transplantation to kidney. In pancreas in situ, most TH-LI cells expressed INS-LI while less than 5% expressed SOM-LI. The total number of TH-LI cells/mm2 was significantly increased directly after isolation and in 0-day, 12-week and 52-week old grafts, but not in 3-day grafts. The proportion of TH-LI cells expressing SOM-LI increased after transplantation, amounting to about one-third by 52 weeks. As expressed per unit islet area, the frequencies of both TH/INS and TH/SOM cells increased significantly in the transplants. The results demonstrate that TH occurs in both beta-cells and D-cells of adult islets. In both cell types the enzyme appears to be responsive to the microenvironmental changes inherent in transplantation. This cellular phenotype plasticity might contribute to the altered insulin secretory dynamics in islet grafts.

Peptides and other neuronal markers in transplanted pancreatic islets.

Functional alterations are developed in transplanted islets over time. Because islets in situ are densely innervated and isolation disconnects the endocrine organ from extrinsic nerves and from ganglia in the exocrine pancreas, it is important to examine the reinnervation of islet grafts. This review describes the patterns of appearances of intrinsic perikarya and reinnervating fibers demonstrating markers for parasympathetic, sympathetic or sensory nerve substances, most notably neuropeptides, in islet transplants. An altered innervation pattern, as compared to normal islets, develops. Presumably the expression of neuronal markers in the grafts is related to factors both in the islets and in the ectopic environment offered by the implantation organ.

Intrinsic and extrinsic NPY nerves in transplanted neuroinsular complexes.

In mouse pancreatic islets, whether in situ or transplanted to kidney, nerve fibers and a few perikarya expressed NPY-like immunoreactivity (NPY-LI). In 4-5 day old grafts, NPY-LI coexisted with VIP-LI in randomly distributed nerve fibers. By 2-52 weeks, NPY mainly co-existed with tyrosine hydroxylase in fibers emanating from the kidney parenchyma. Radioimmunoassays indicated that the NPY levels increased with time, while those of VIP decreased. The study shows that NPY is primarily present in the intrinsic VIP-ergic innervation of islet grafts but later is mainly a constituent of the ingrowing sympathetic innervation.

Expression of tyrosine hydroxylase, calcitonin gene-related peptide, substance P and protein gene product 9.5 in mouse islets transplanted under the kidney capsule.

Pancreatic islets transplanted to the kidney of syngeneic mice were stained for calcitonin gene-related peptide (CGRP), substance P (SP), tyrosine hydroxylase (TH), acetylcholinesterase and the pan-neuronal marker, protein gene product 9.5 (PGP). Nerve fibers expressing TH-like immunoreactivity (TH-LI) and CGRP-LI were rare for 4 days but increased 2 (CGRP) or 6 (TH) weeks after transplantation. In 1-year-old grafts the CGRP-LI innervation resembled that in situ, while TH-LI and PGP-LI innervations were increased. SP-LI fibers remained rare throughout. Perikarya intrinsic to the islets did not show CGRP-LI or SP-LI. The results indicate a progressive ingrowth of sensory fibers into the grafts and that the TH-LI innervation becomes even more pronounced than in the pancreas. The post-transplantation reaction of islet intrinsic neurons does not involve CGRP and SP, contrasting with previous observations for vasoactive intestinal polypeptide.

Neuroinsular complex type I: morphology and frequency in lean and genetically obese mice.

No quantitative data are available regarding the rate of occurrence of nerve cells in association with endocrine pancreas (i.e.. neuroinsular complexes type I [NICs]), or the difference in the distribution of NICs in normal and diabetic pancreas. In this report, pancreata from 20-day, 7-week, and 9-month-old lean (Umeå +/?) and obese (Umeå ob/ob) mice, as well as 10-month-old C57BL/6JBom and Umeå ob/ob mice, were analyzed with regard to the association of acetylcholinesterase (AChE)-positive and protein gene product 9.5-like (PGP-LI) immunoreactive perikarya with islets, and not in association with islets. NIC profiles were regularly observed, but were more frequent in the 20-day-old mice than in the 9-month-old +/? and ob/ob mice. The NIC profiles were often located close to a duct or blood vessel, significantly more frequently than islet profiles in general. The data did not reveal any gross abnormality in ob/ob mice as regards the frequency of NICs or the number of AChE-positive and PGP-LI perikarya. However, the 9-month-old ob/ob mice demonstrated smaller clusters of perikarya in their NIC profiles as compared to the other mice, probably reflecting the fact that the perikarya were more widely spread out in the hyperplastic islets of adult ob/ob mice. The results show that NICs are common and represent a substantial proportion of the islets in mouse pancreas, supporting the idea that they play a role in islet physiology.

Mouse islets cultured with vasoactive intestinal polypeptide: effects on insulin release and immunoreactivity for tyrosine hydroxylase.

Mouse islets cultured for 1 or 4 days with or without 10 nM vasoactive intestinal polypeptide (VIP) were stained for tyrosine hydroxylase (TH) and examined for insulin secretion during culture and in a postculture perifusion system. Exposure to exogenous VIP for 4 days increased the frequency of islet cells expressing TH-like immunoreactivity. Regardless of the culturing conditions, the islets exhibited significant insulin secretory responses to 16.7 mM glucose, the effect being potentiated by 10 nM VIP in the perifusion medium. The insulin-releasing action of glucose and the potentiating effect of VIP were less pronounced in islets cultured for 1 day with VIP than in islets cultured without this neuropeptide. The following conclusions are suggested: (a) VIP stimulates the expression of TH in mouse islet cells; (b) the latency of the VIP-induced TH is a postreceptor phenomenon; (c) islet cultures exposed to VIP represent a new instance of the association between increased functional demands on beta cells and enhanced expression of TH and a new instance of VIP having trophic effects.

Effects of leptin, acetylcholine and vasoactive intestinal polypeptide on insulin secretion in isolated ob/ob mouse pancreatic islets.

Obesity is often accompanied by hyperleptinemia, hyperinsulinemia, and an increased parasympathetic tone. Obese-hyperglycemic mice (Umeå ob/ob) have functional leptin receptors and a raised parasympathetic tone. We studied insulin release in islets isolated from 9-month-old severely obese ob/ob mice. Leptin (0.5-18 nM) did not affect insulin release together with 2.8-20 mM glucose. Leptin (18 microM) had no effect in the presence of low glucose (2.8-5.5 mM), but increased insulin secretion in islets challenged with 11.1 or 16.7 mM glucose. Leptin at 18 microM increased insulin secretion stimulated by the parasympathetic neurotransmitters acetylcholine (ACh; 10 microM) or vasoactive intestinal peptide (VIP; 10 nM), and by 5 mM theophylline or 2.5 microM forskolin. Overnight culture increased the effect of 18 microM leptin, but no effects were observed with 18 nM leptin. Pretreatment of islets with phorbol 12-myristate 13-acetate (PMA) did not suggest any involvement of protein kinase C. In summary, a high concentration of leptin stimulates insulin release in the presence of stimulatory concentrations of glucose alone and with parasympathetic neurotransmitters. Hyperleptinemia and increased parasympathetic stimulation may in part cause the hyperinsulinemia observed in obesity. This may aggravate insulin resistance and the abnormal metabolism in diabetes mellitus.

Effects of bone wax on rabbit cranial bone lesions.

The present study was designed to elucidate the reactions of cranial membranous bone to bone wax. In ten young rabbits, twenty parietal bone defects were created by drilling, the edges of which were partly extended using rongeur forceps to enable investigation of eventual thermal effects. Half of the marginal bone surrounding the lesions was covered by bone wax, the remainder serving as control. The animals were sacrificed 1 and 7 weeks after surgery, and block specimens prepared for light microscopy. Merely slight tissue reactions to the bone wax were discerned. Bony regeneration occurred mainly from the dura mater and the pericranium, but also from the bony rim. Reskeletalization was markedly impaired by the presence of bone wax. Heat generated by drilling caused reduced bone formation despite constant irrigation peroperatively. Clinical consequences are discussed.

Regeneration of cranial suture and bone plate lesions in rabbits. Implications for positioning of osteotomies.

The positioning of osteotomies in intramembranous cranial bone was studied by exploring the pattern of bone regeneration in growth areas (the sutural region) as compared to that of the bone plate proper. Trephine defects in the left coronal suture area and the right parietal bone were produced in fifty-nine young rabbits. A pilot study to refine operative and analytical methods comprised 22 animals. The experiments were terminated at one, three, and six weeks after surgery. The bone regenerative response was assessed by x-ray planimetry, plain microscopy, enzyme histochemistry, and fluorescent labelling. Only minor divergences in healing capacity between the two defects were found. No adverse effects on the growth process were indicated. As to clinical management, the findings suggest that osteotomies designed to traverse sutural areas will, under normal circumstances, regenerate in a similar manner and rate to adjoining bone plates.