The effect of parotid gland-sparing intensity-modulated radiotherapy on salivary composition, flow rate and xerostomia measures.

OBJECTIVES: To describe parotid gland (PG) saliva organic and inorganic composition and flow rate changes, after curative intensity-modulated radiotherapy (IMRT) for head and neck cancer (HNC), and analyse the relationship between PG saliva analytes and xerostomia measures. METHODS AND MATERIALS: Twenty-six patients recruited to five prospective phase 2 or 3 trials which assessed toxicity and efficacy of IMRT by HNC subsite, provided longitudinal PG saliva. Salivary flow rate, and subjective and objective xerostomia measures were prospectively collected and saliva tested for inorganic and organic analytes. Statistical comparisons of longitudinal analyte changes and analysis for a relationship between dichotomized xerostomia score and saliva analytes were performed. RESULTS: One hundred and forty-two PG saliva samples from 26 patients were analysed. At 3-6 months after IMRT, stimulated and unstimulated saliva showed significantly decreased flow rate, total protein (TP) secretion rate, phosphate concentration and increased lactoferrin (LF) concentration. Stimulated saliva alone had elevated LF secretion rate and beta-2-microglobulin (B2 M) concentration with decreased calcium (Ca2+ ) and magnesium (Mg2+ ) concentrations and Ca2+ secretion rate. At >12 months, under stimulated and unstimulated conditions, increased LF concentration and decreased Mg2+ and phosphate concentration persisted and, in stimulated saliva, there was decreased potassium (K+ ) and Mg2+ concentration. Unstimulated TP secretion rate was lower in the presence of high-grade xerostomia. Otherwise, no relationship between xerostomia grade and PG salivary flow rate, TP and Ca2+ secretion rate was found. CONCLUSION: Fewer significant differences in PG saliva analytes >12 months after IMRT indicate good functional recovery. Residual xerostomia after IMRT will only be further reduced by addressing the sparing of subsites of the PG or other salivary gland tissues, in addition to the PG.

Intensity modulated radiotherapy in locally advanced thyroid cancer: Outcomes of a sequential phase I dose-escalation study.

BACKGROUND AND PURPOSE: To determine the safety and tolerability of dose-escalation using modestly accelerated IMRT in high-risk locally advanced thyroid cancer requiring post-operative radiotherapy, and to report preliminary data on efficacy. MATERIALS AND METHODS: A sequential Phase I dose-escalation design was used. Dose level one (DL1) received 58.8 Gy/28F to the post-operative bed and 50 Gy/28F to elective nodes. DL2 received 66.6 Gy/30F to the thyroid bed, 60 Gy/30F to post-operative nodal levels and 54 Gy/30F to elective nodal levels. Acute (NCICTCv.2.0) and late toxicities (RTOG and modified LENTSOM) were recorded. The primary endpoint was the number of patients with ≥Grade 3 (G3) toxicity at 12 months post-treatment. RESULTS: Fifteen patients were recruited to DL1 and twenty-nine to DL2. At 12 months ≥G3 toxicities were 8.3% in both DL1 and DL2. At 60 months, ≥G3 toxicity was reported in 3 (33%) patients in DL1 and 1 (7%) in DL2. One patient in DL2 died at 24 months from radiation-induced toxicity. Time to relapse and overall survival rates were higher in DL2, but this was not statistically significant. Dose-escalation using this accelerated regimen can be safely performed with a toxicity profile similar to reported series using conventional doses.

Inverse regulation of cyclin B1 by c-Myc and p53 and induction of tetraploidy by cyclin B1 overexpression.

We have shown previously that mitotic spindle inhibitors allow the c-Myconcoprotein to uncouple mitosis from DNA synthesis, resulting in the acquisition of tetraploidy. This can also occur in the absence of spindle inhibition if c-Myc deregulation is combined with inactivation of the p53 tumor suppressor. Under these conditions, cyclin B1 protein is induced but retains its normal cell cycle regulation. We now show that the cyclin B1 promoter is directly but oppositely regulated by c-Myc and p53. Enforced expression of cyclin B1 also induces tetraploidy, either after mitotic spindle inhibition or in the absence of such inhibition if cyclin B1 is coexpressed with c-Myc. Cyclin B1 represents a new class of c-Myc target genes that is also regulated by p53. It is also the first identified downstream effector of c-Myc able to produce the chromosomal instability that characterizes virtually all tumor cells.

Total Mucosal Irradiation with Intensity-modulated Radiotherapy in Patients with Head and Neck Carcinoma of Unknown Primary: A Pooled Analysis of Two Prospective Studies.

AIMS: To determine the clinical outcomes of an intensity-modulated radiotherapy technique for total mucosal irradiation (TM-IMRT) in patients with head and neck carcinoma of unknown primary (HNCUP). MATERIALS AND METHODS: A single-centre prospective phase II trial design was used in two sequential studies to evaluate TM-IMRT for HNCUP. Patients were investigated for primary tumour site using examination under anaesthetic and biopsies, computed tomography ± magnetic resonance imaging (MRI) or 18-fluorodeoxyglucose positron emission tomography-computed tomography (PET-CT). Patients received IMRT to the potential primary tumour sites and elective cervical nodes. Concomitant chemotherapy was used in patients who received primary radiotherapy or those with nodal extracapsular extension. RESULTS: Thirty-six patients with HNCUP were recruited; 72% male. Twenty-five patients (69.4%) had p16-positive disease. Two year mucosal and local nodal control rates were 97.1% (95% confidence interval 91.4-100) and 89.8% (78.4-100), respectively. One mucosal primary was detected 7.3 months after TM-IMRT and three patients died from recurrent/metastatic squamous cell carcinoma of the head and neck. Twelve patients (33%) developed grade 3 (Late Effects in Normal Tissue-Subjective, Objective, Management and Analytical; LENT-SOMA) dysphagia with a 1 year enteric tube feeding rate of 2.7%. The high-grade subjective xerostomia rate (LENT-SOMA) at 24 months after IMRT was 15%. CONCLUSIONS: At a median follow-up of 36.1 months, the use of TM-IMRT was associated with good local control. Toxicity was comparable with previously reported TM-IMRT regimens encompassing similar mucosal volumes.

Lack of transcriptional repression by max homodimers.

Max, a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) protein, plays a central role in the transcriptional regulation of myc oncoprotein-responsive genes. Myc-max heterodimers bind to consensus E-box motifs near or within the promoters of these genes and activate gene expression, whereas heterodimers between max and members of the mad family of bHLH-ZIP proteins promote transcriptional repression. In contrast to all other members of the myc network, max readily homodimerizes and binds to identical E-box sites in vitro. However, the role for max homodimers in transcriptional repression in vivo is unclear. Upstream stimulatory factor (USF) is a bHLH-ZIP protein which does not interact with members of the myc-max-mad family. By replacing the HLH-ZIP domain of max with that from USF, we created a chimeric protein, max(USF), which was indistinguishable from max with respect to its ability to homodimerize and bind DNA. As expected, however, max(USF) was unable to heterodimerize with any of the tested max partner proteins and was incapable of suppressing c-myc target genes. Thus, transcriptional repression is an exclusive property of max-mad heterodimers and cannot be achieved by max homodimers alone.

Mmip1: a novel leucine zipper protein that reverses the suppressive effects of Mad family members on c-myc.

C-myc, a member of the basic helix-loop-helix-leucine zipper (bHLH-ZIP) protein family activates target genes in heterodimeric association with another bHLH-ZIP protein, Max. Max readily homodimerizes, competes with C-myc-Max heterodimers, and represses transcription. Four additional bHLH-ZIP proteins, Mad1, Mxi1, Mad3 and Mad4, heterodimerize with Max and also repress transcription of c-myc-responsive genes. We employed a yeast two-hybid approach to identify proteins which interact with Mxi. We identified a novel ZIP-containing protein, Mmip1 (Mad member-interacting protein 1) that strongly dimerizes with all four Mad members, but not with c-myc, Max, or with unrelated HLH proteins. The Mmip1-Mxi association is mediated by the ZIP domain of each polypeptide and is as strong or stronger than the associations between c-myc and Max or Max and Mxi1. In vitro, Mmip1 can inhibit DNA binding by Max-Mad heterodimers and, in vivo, can reverse the suppressive effects of Mad proteins on c-myc functions. Mmipl is found in a variety of cells types, is induced by serum stimulation, and can be co-immunoprecipitated from fibroblasts in association with Mxi1. By interfering with the dimerization between Max and Mad family member proteins, Mmip1 can indirectly up-regulate the transcriptional activity of c-myc and suppress the antiproliferative actions of Mad proteins.

Mmip-2/Rnf-17 enhances c-Myc function and regulates some target genes in common with glucocorticoid hormones.

Members of the Mad family of basic-helix-loop-helix-leucine zipper proteins inhibit the transcriptional activity of the c-Myc oncoprotein. Mmip-2/Rnf-17 is a RING-finger protein that interacts with all four known Mad proteins, redistributes them to the cytoplasm, and thus enhances c-Myc function. We generated cell lines in which Mmip-2/Rnf-17 was rendered glucocorticoid (GC)-inducible. Stable expression of Mmip-/Rnf-17 resulted in the expected transport of the most abundant endogenous mad protein, Mxi1, to the cytoplasm. Compensatory increases in Mxi1 and Mad3 transcripts, similar to those previously described in Mad1 null hematopoietic cells, were also seen. Mmip-2/Rnf-17 also sensitized cells to several different pro-apoptotic stimuli and regulated a subset of c-Myc target genes. Unexpectedly, some of these genes were also found to be modulated solely by GCs. Thus, the inhibition of Mad proteins by Mmip-2/Rnf-17 modulates c-Myc function by enhancing its ability to regulate a subset of its potential target genes. Our results also identify a previously unrecognized overlap between genes regulated by c-Myc- and GCs and provide a potential molecular basis for their regulation of common cellular functions.

Genetic dissection of c-myc apoptotic pathways.

All biological functions mediated by the c-myc oncoprotein require an intact transactivation domain (TAD). We compared TAD mutants for their ability to promote apoptosis of 32D myeloid cells in response to interleukin-3 (IL-3) deprivation and exposure to chemotherapeutic drugs, and to activate ornithine decarboxylase, an endogenous c-myc target. Different sub-regions of the TAD were required to mediate each function. cDNA microarrays were then used to identify multiple c-myc-regulated transcripts, some of which were also modulated by IL-3 or cytotoxic drugs, as well as by specific sub-regions of the TAD. Several of the c-myc-regulated transcripts had also been previously identified as targets for IFN-gamma. The functional consequences of their deregulation were manifested by a marked sensitivity of c-myc-overexpressing cells to IFN-gamma-mediated apoptosis. Our results establish that several well-characterized functions of c-myc are separable and correlate with the expression of a novel group of target genes, some of which also mediate the apoptotic action of IFN-gamma.

C-myc overexpression and p53 loss cooperate to promote genomic instability.

p53 monitors genomic integrity at the G1 and G2/M cell cycle checkpoints. Cells lacking p53 may show gene amplification as well as the polyploidy or aneuploidy typical of many tumors. The pathways through which this develops, however, are not well defined. We demonstrate here that the combination of p53 inactivation and c-myc overexpression in diploid cells markedly accelerates the spontaneous development of tetraploidy. This is not seen with either N-myc or L-myc. Tetraploidy is accompanied by significantly higher levels of cyclin B and its associated cdc2 kinase activity. Mitotic spindle poisons accelerate the appearance of tetraploidy in cells either lacking functional p53 or overexpressing c-myc whereas the combination is additive. Restoration of p53 function in cells overexpressing c-myc causing rapid apoptosis, indicating that cells yet to become tetraploid have nonetheless suffered irreversible genomic and/or mitotic spindle damage. In the face of normal p53 function, such damage would either be repaired or trigger apoptotis. We propose that loss of p53 and overexpression of c-myc permits the emergence and survival of cells with increasingly severe damage and the eventual development of tetraploidy.

Promotion of growth and apoptosis in c-myc nullizygous fibroblasts by other members of the myc oncoprotein family.

c-myc nullizygous fibroblasts (KO cells) were used to compare the abilities of c-myc, N-myc and L-myc oncoproteins to accelerate growth, promote apoptosis, revert morphology, and regulate the expression of previously described c-myc target genes. All three myc oncoproteins were expressed following retroviral transduction of KO cells. The proteins all enhanced the growth rate of KO cells and significantly shortened the cell cycle transition time. They also accelerated apoptosis following serum deprivation, reverted the abnormal KO cell morphology, and modulated the expression of previously described c-myc target genes. In most cases, L-myc was equivalent to c-myc and N-myc in restoring all of the c-myc-dependent activities. These findings contrast with the previously reported weak transforming and transactivating properties of L-myc. Myc oncoproteins may thus impart both highly similar as well as dissimilar signals to the cells in which they are expressed.

Effects of d-amphetamine and diazepam on paired and grouped primate food competition.

Two male and two female rhesus monkeys (Macaca mulatta) were the subjects of an experiment designed to assess the effect of d-amphetamine (DA; 0.125, 0.5 and 2.0 mg/kg, IM) and diazepam (DZP; 0.5 and 2.5 mg/kg, IM) on food-getting behavior in paired and group competition. Paired competition results show that in some cases submissive animals, that had previously failed to obtain apple pieces, were successful in obtaining some apple pieces when either the dominant animal of the pair or both subjects were given 0.5 mg/kg DA or 2.5 mg/kg DZP. Results revealed the same effect when all animals (group competition) were given 0.125 and 2.0 mg/kg DA and 2.5 mg/kg DZP. These results appear to indicate that the effect of drugs on food-getting behavior in competitive situations is in some manner influenced by the social status of the animal.

Benzhexol and side effects with long-acting fluphenazine therapy.

Sixteen patients who had for some months been on fluphenazine enanthate injections (1-3 ml) every two or three weeks, with daily oral benzhexol (6-40 mg), were divided into two groups comparable in age, diagnostic category, and drug dose.Placebo was substituted under blind controlled conditions for benzhexol in one group, and both groups were regularly assessed by Simpson rating scale for extra-pyramidal signs, and by a 30-question symptom check list. Both assessments were found reliable.Four out of eight patients had severe reactions when off benzhexol, but symptoms such as tremor and daytime sleepiness were unaltered in all patients. The other four did not suffer adversely from withdrawal of benzhexol.Acute withdrawal is therefore unjustified, but occasional revision of dosage of anti-Parkinsonian drugs is advisable. There is no evidence that tolerance develops to any effect of a phenothiazine, but long-continued benzhexol might induce a denervation supersensitivity. It is also possible that Parkinsonian signs are dependent on affective state.

Effects of receptor blockade on metabolism and renal actions of vasopressin in conscious dogs.

Vasopressin--but not the V2 receptor agonist [deamino-cis1,D-Arg8]-vasopressin (dDAVP)--may mediate natriuresis in dogs. The present study investigated this phenomenon by use of nonpeptide antagonists to V1a and V2 receptors 1-¿1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl¿-3,4-dihydro-2 (1H)-quinolinone (OPC-21268) and 5-dimethylamino-1-¿4-(2-methylbenzoylamino)-benzoyl¿-2,3,4,5-tetra hydro-1 H-benzazepine (OPC-31260), respectively) hypothesising that only V1a inhibition would reduce the natriuresis. In conscious dogs vasopressin secretion was suppressed by water loading (2% body weight) and replaced by infusion of vasopressin (50 pg min-1 kg-1) resulting in physiological plasma concentrations (plasma levels of AVP (pAVP) = 2.0 +/- 0.1 pg mL-1). In this setting, OPC-21268 did not change the rate of sodium excretion. OPC-31260 increased water excretion 12-fold without significant changes in sodium excretion. Heart rate, mean arterial blood pressure, glomerular filtration rate, and clearance of endogenous Li+ were unchanged. During vasopressin infusion, both antagonists increased pAVP, OPC-21268 by 20% and OPC-31260 by 100% (2.0 +/- 0.2-4.0 +/- 0.3 pg mL-1). In the absence of vasopressin infusion, OPC-31260 did not increase pAVP. Thus, the increase in pAVP appeared to be due to a decrease in metabolic clearance rate. The results indicate that the present dose of V1a receptor inhibitor OPC-21268 does not reduce sodium excretion and that both vasopressin antagonists inhibit vasopressin metabolism.

Behavioral effects of fetal and neonatal exposure to methadone in the rat.

Twenty-two pregnant Sprague-Dawley rats were maintained on methadone hydrochloride solution (14 mg/kg/day, PO, ad lib) throughout gestation, or served as prenatal controls with equal intake of tap water. Within 48 hr of birth 129 pups were cross-fostered to dams of postnatal control or methadone (27 mg/kg/day, PO, ad lib) status. A significant increase in stillbirths occurred among prenatal methadone litters. Disturbed maternal behavior sometimes developed when a dam was switched from prenatal control to postnatal methadone or vice versa. Foster pups of such females showed long-lasting growth retardation and less emotionality in Open Field trials as adults. They were less active on Day 28 in a circular path test, but when tested after Day 65, they were more active. Pups exposed to methadone both pre- and post-natally were lest active in initial Open Field tests (both on Day 28 and after Day 65) of all treatment groups. No severe withdrawal symptoms were noted in pups at birth or weaning. The effect of methadone on growth, emotionality and activity level was mild compared to the effects of drug-induced maternal disorganization.

Novel regulation of the helix-loop-helix protein Id1 by S5a, a subunit of the 26 S proteasome.

Id proteins negatively regulate the dimerization, DNA binding, and biological properties of basic helix-loop-helix proteins. In a search for novel factors that interact with Id1, we identified a component of the 26 S proteasome, S5a, that has previously been implicated only in the recognition of ubiquitinated polypeptides destined for proteolysis. S5a interacts strongly with Id1, less strongly with the basic helix-loop-helix proteins MyoD and E12, and not at all with other Id proteins. S5a restores DNA binding by MyoD-Id1 and E12-Id1 heterodimers, enhances DNA binding by MyoD and E12 homodimers, and reverses Id1-mediated repression of the muscle creatine kinase promoter during myogenic differentiation. Mutagenesis experiments showed that amino acids flanking the helix-loop-helix domain plus three residues in the first helix of Id1 impart S5a recognition. This requires only the NH2-terminal half of S5a. S5a thus appears to promote the positive regulation of myogenic genes through ubiquitin-independent mechanisms involving inhibition of Id1 and the enhancement of DNA binding by MyoD and E12. This latter property may permit the selection of novel promoter binding sites during myogenesis.

Differential apoptotic behaviors of c-myc, N-myc, and L-myc oncoproteins.

c-, N-, and L-myc are related nuclear oncoproteins that bind similar DNA sites and cooperate with activated ras oncogenes to transform primary fibroblasts. Although c-myc can also promote apoptosis in some cells after growth factor withdrawal or exposure to cytotoxic agents, roles for N- and L-myc in apoptosis remain undetermined. To address this, c-, N-, or L-myc were stably expressed in the interleukin 3 (IL-3)-dependent 32D hematopoietic cell line. The apoptotic response of each cell line was assessed after IL-3 withdrawal or treatment with four structurally unrelated cytotoxic agents. All three oncoproteins accelerated apoptosis after IL-3 withdrawal. In contrast, whereas c-myc overexpression generally sensitized cells to cytotoxic drugs, N-myc and L-myc overexpression produced resistance. myc expression tended to be associated with a more robust G2-M arrest after drug exposure, but this did not correlate with drug sensitivity or resistance. Bcl-2 and Bcl-X(L) protected control cells against apoptosis after either IL-3 withdrawal or drug exposure, although in some cases this effect could be overridden by myc oncoproteins, particularly N-myc and L-myc. Our results suggest that the apoptotic pathways activated upon IL-3 withdrawal and cytotoxic drug treatment are distinct and differentially affected by members of the myc and Bcl-2 families.

Mechanism of vasopressin natriuresis in the dog: role of vasopressin receptors and prostaglandins.

Renal effects of physiological amounts of vasopressin were studied in conscious dogs during servocontrolled overhydration (2% body wt). During infusion of vasopressin (50 pg . min-1 . kg body wt-1), plasma vasopressin concentration increased to 2.30 +/- 0.20 pg/ml compared with 0.12 +/- 0.03 pg/ml during control (water diuresis). With vasopressin infusion, urine flow was significantly lower (0.30 +/- 0.10 ml/min) and sodium excretion (UNaV) was significantly higher (58.0 +/- 15.8 micromol/min) than without vasopressin (4.6 +/- 0.4 ml/min and 14.4 +/- 4.1 micromol/min, respectively). Deamino-[Cys1,D-Arg8]vasopressin, a V2 receptor agonist (4 pg . min-1 . kg-1), mimicked the antidiuretic response (0.20 +/- 0.03 ml/min) without changing UNaV (9.7 +/- 4.4 micromol/min). Indomethacin given during arginine vasopressin (AVP) infusion suppressed prostaglandin E2 excretion, intensified the antidiuresis (0.10 +/- 0.02 ml/min), and abolished the natriuresis (13.4 +/- 3.7 micromol/min). During AVP infusion, UNaV was highly correlated (r = 0.85) with prostaglandin E2 excretion. Blood pressure, glomerular filtration rate, plasma atrial natriuretic peptide concentration, and the rate of proximal tubule reabsorption (derived from lithium clearance) were similar in all series. The data indicate that, in the dog, physiological amounts of vasopressin can induce natriuresis, probably through activation of non-V2 receptors and the intrarenal synthesis of prostaglandins.

An evaluation of automated defibrillation and manual defibrillation by emergency medical technicians in a rural setting.

We show that automated external defibrillation training of emergency medical technicians (EMTs) is less time consuming than manual defibrillation training, and hypothesize that both improve survival from sudden cardiac death. Data on 91 cardiac arrests over 27 months among five basic life support services was collected before EMT-defibrillation (EMT-D) training. Subsequently, seven BLS services were trained in EMT-D using either manual difibrillation or automated external defibrillation technology, and 55 sudden cardiac death patients were entered after training. Manual defibrillation required 11 more hours per student in initial training. Survival to hospital discharge improved from two of 91 patients (2.2%) in the series before EMT-D training to nine of 55 patients (16.4%) after EMT-D training (P = .001). Improved survival was correlated with shorter prehospital defibrillation times, 8.84 minutes, when EMTs performed defibrillation versus 16.3 minutes before training when EMTs awaited advanced life support defibrillation (P < .001). To enhance equipment familiarity we allowed EMTs to apply three-lead electrode monitors to all medical/cardiac patients during transport (surveillance). There were six emergency medical service-witnessed "surveillance" arrests and three arrests survived to hospital discharge (50% survival). This group represented 33% of all survivors in the series. We recommend automated external defibrillation training for EMTs. Improved survival in sudden cardiac death cases in well-run emergency medical service systems should result from EMT-D training. Finally, we recommend that routine "surveillance" of high-risk patients during transport by defibrillation-capable EMTs be considered in EMT-D programs, rather than limiting EMT-D only to units capable of rapid "man-down" response.

Commonly occurring loss and mutation of the MXI1 gene in prostate cancer.

One of the most common chromosomal abnormalities in prostate cancer involves loss of 10q22-qter. Rarely, a smaller deletion, involving 10q24-q25, has been observed, suggesting the presence of a tumor suppressor gene at this site. We previously demonstrated that the MXI1 gene maps to 10q24-q25 and is mutated in some tumors with cytogenetically detectable deletions of this locus. MXI1 encodes a basic-helix-loop-helix protein that suppresses the transcriptional activity of the MYC oncoprotein by competing for the common dimerization partner, MAX, and binding to identical DNA sites. Because more than 90% of prostate tumors contain no cytogenetic abnormality of 10q, the relevance of MXI1 loss and/or mutation to the vast majority of cases remains unclear. We prospectively evaluated prostate tumors for loss of MXI1 by fluorescence in situ hybridization (FISH) and cytogenetic techniques. Twenty-one of 40 tumors (53%) demonstrated loss of a single MXI1 allele as determined by FISH. Ten cases with cytogenetically normal 10qs, but with FISH-documented deletion of MXI1, were examined at the molecular level, and eight mutations were identified, albeit at low frequency. Five of the mutant proteins were unable to bind DNA in association with MAX. We conclude that MXI1 gene loss in prostate cancer is common and most frequently involves a cytogenetically undetectable deletion.