The DNA-binding domain of yeast heat shock transcription factor independently regulates both the N- and C-terminal activation domains.

The expression of heat shock proteins in response to cellular stresses is dependent on the activity of the heat shock transcription factor (HSF). In yeast, HSF is constitutively bound to DNA; however, the mitigation of negative regulation in response to stress dramatically increases transcriptional activity. Through alanine-scanning mutagenesis of the surface residues of the DNA-binding domain, we have identified a large number of mutants with increased transcriptional activity. Six of the strongest mutations were selected for detailed study. Our studies suggest that the DNA-binding domain is involved in the negative regulation of both the N-terminal and C-terminal activation domains of HSF. These mutations do not significantly affect DNA binding. Circular dichroism analysis suggests that a subset of the mutants may have altered secondary structure, whereas a different subset has decreased thermal stability. Our findings suggest that the regulation of HSF transcriptional activity (under both constitutive and stressed conditions) may be partially dependent on the local topology of the DNA-binding domain. In addition, the DNA-binding domain may mediate key interactions with ancillary factors and/or other intramolecular regulatory regions in order to modulate the complex regulation of HSF's transcriptional activity.

The synthesis and evaluation of temperature sensitive tubulin toxins.

The synthesis of several potent inhibitors of tubulin polymerization that exert their activities through interaction at the colchicine binding site is described. These agents were evaluated for their abilities to inhibit the polymerization of tubulin and the growth of neoplastic cell cultures. Additionally, the inhibition of tubulin polymerization activity of these agents was assessed over a temperature range of 30-45 degrees C to ascertain the effect of temperature on this activity. Several of the compounds possess significant inhibition of tubulin polymerization activity, and select compounds exhibit this activity in a temperature dependent manner.

The synthesis and evaluation of benzannelated-azatoxins: the benzazatoxins.

The synthesis and evaluation of azatoxin congeners possessing annealed aromatic frameworks are described. The compounds were evaluated for their abilities to affect topoisomerase II inhibition through the stabilization of "cleavable complex" and for the inhibition of tubulin polymerization using purified bovine brain tubulin.

Synthesis and structure-activity profiles of A-homoestranes, the estratropones.

2-Methoxyestradiol, a mammalian metabolite of estradiol, has reported antiangiogenic activity which has been proposed to be mediated through interaction at the colchicine binding site on the tubulin monomer. Subsequent structure-activity studies of 2-methoxyestradiol have yielded highly potent steroidal inhibitors of tubulin polymerization. In an effort to probe the scope of binding at the colchicine binding site and the nature of the relationship between 2-methoxyestradiol and colchicine, a series of colchicine/2-methoxyestradiol hybrids was synthesized. These A-homoestrane hybrid systems, collectively termed estratropones, possessed an A-ring tropone system with the keto functionality at either the C-2, C-3, or C-4 position of the steroid nucleus. The estratropones were evaluated for their ability to inhibit the polymerization of tubulin using an in vitro purified bovine brain assay. Most of these hybrids inhibit polymerization with greater potency than either of the natural products. The most potent of these congeners possessed an approximate 5-fold enhancement of the activity of colchicine for the inhibition of tubulin polymerization. alpha-Substituents on the tropone ring showed varied effects on the activities for the two classes of estratropones studied in this regard, the C-3 oxo and the C-4 oxo species. The 3-substituted 4-oxoestratropones exhibited antitubulin activity according to Cl approximately Br > OCH3, whereas the 4-substituted 3-oxoestratropones exhibited activity according to OCH3 > Br approximately Cl. It is unclear if these substituent factors are purely electronic or steric effects or if the substituent operates indirectly by altering the conformation of the nonplanar troponoid ring. The estratropones represent a new class of tubulin binding agents with potential antiangiogenic utility.

Sunburn and tanning in a British population.

BACKGROUND: The prevalence of sunburn and tanning, and associated attitudes were investigated in a national sample of adults in Great Britain. METHODS: A randomly selected cross-sectional sample of 2025 adults aged 16 or over living in England, Scotland and Wales were interviewed in October 1993 in the Office of Population Censuses and Surveys Omnibus Survey. RESULTS: The response rate was 77 per cent. Thirty-seven per cent reported at least one episode of sunburn in the past 12 months, the highest frequencies being found in young age groups, in those with skin that tans poorly (p < 0.001), and in those who specifically tried to tan in this period (p < 0.001). Sunbathing was the most frequent activity associated with most severe episodes of sunburn, and occurred most frequently at the beginning of the summer, 46 per cent of episodes occurring abroad. Thirty-three per cent had tried to tan in the past 12 months, the proportion being highest in younger age groups and women (p < 0.001). Among those with severe sunburn more men (34 per cent) expected to burn when trying to tan than women (17 per cent). CONCLUSIONS: The results provide background information with which to develop and evaluate primary prevention initiatives for skin cancer. These should take into account the strong association between sunburn and the desire to tan, particularly in young adults and men.

Physiological effect of endobronchial radiotherapy in patients with major airway occlusion by carcinoma.

BACKGROUND: Endobronchial radiotherapy by a high dose rate remote after-loading technique (high dose rate brachytherapy) has become an established treatment for major airway occlusion by inoperable carcinoma of the bronchus. Only limited objective data on its effect on pulmonary physiology and on radiographic and bronchoscopic appearances are available. The aim of this study was to make a detailed assessment of patients before and after high dose rate brachytherapy to determine which investigations were useful and to generate data for comparing this with other methods of treatment. METHODS: Twenty patients with major airway obstruction by inoperable lung cancer underwent a detailed assessment before receiving endobronchial radiotherapy (15 Gy at 1 cm in a single fraction) and six weeks after treatment. This included chest radiography, computed tomography of the thorax, bronchoscopy including an obstruction index, five minute walking tests, isotope ventilation and perfusion lung scanning, and full lung function tests with maximum inspiratory and expiratory flow-volume loops. RESULTS: Nineteen patients (mean age 69 years) completed the study. Symptomatic improvement occurred in 17 patients. A collapsed lobe or lung, seen on the chest radiograph in 13, reexpanded in nine. Bronchoscopic appearances improved in 18, the mean obstruction index decreasing from 6.2 to 2.8. The isotope scans showed significant increases in the percentage of total lung ventilation (V) and perfusion (Q) measured over the abnormal lung (V 17.7% to 27.7%, Q 15.1 to 21.9%). Five minute walking distance (305 to 329 m), forced expiratory volume in one second (FEV1 1.45 to 1.61 l), forced vital capacity (FVC 2.17 to 2.48 l) and ratio of forced expiratory to forced inspiratory flow rate at 50% vital capacity (FEF50/FIF50 0.58 to 0.88) all increased significantly. CONCLUSIONS: Endobronchial radiotherapy led to subjective benefit in most cases in terms of symptoms and bronchoscopic and radiological appearances. There was objective improvement in spirometric indices and in exercise tolerance with increased pulmonary ventilation and perfusion and evidence of decreased intrathoracic airway obstruction.

The use of discriminant analysis to guide palliative treatment for lung cancer patients.

The aim of this study was to develop a prognostic index for patients with inoperable non-small cell lung cancer which could predict survival to 3 months. This would enable less radiation dose to be given to patients where prognosis is limited by occult metastases, giving rise to less treatment morbidity and raising the therapeutic ratio. Data on 18 known prognostic factors were collected on 96 patients. Performance status, lymphocyte count, weight loss and extent of disease were the most predictive factors and were combined into an index. Logistic discriminant analysis was employed to give a numerical score of likelihood of survival to 3 months, ranging from 0 (not likely) to 1 (certain). In this first set of 96 patients, 16 deaths were observed before 3 months, of which 6 were predicted. There was one false positive prediction. Overall accuracy of prediction was therefore 89% with 99% specificity. The same 4 prognostic factors were measured on a second set of 80 patients. Nineteen died before 3 months, of which 5 were predicted with 2 false positives, giving an overall accuracy of 80% and 97% specificity. A probability of survival of less than or equal to 0.2, although highly specific, was only applicable to 9% of patients and this was the limiting factor in the clinical usefulness of the test. A 16-branch tree diagram allows any patient to be assigned a risk factor based on the four predictive factors at the first clinic attendance. Use of the index could encourage more rational prescribing of radiation dose.

Long term results of Cathetron high dose rate intracavitary radiotherapy in the treatment of carcinoma of the cervix.

Between 1967 and 1974, 371 patients with carcinoma of the cervix have been treated by a combination of external beam radiotherapy and fractionated high dose rate brachytherapy using the Cathetron. A retrospective review was undertaken in 1986 and median follow-up time was 6 years. Life table analysis of survival and complications to 16 years was undertaken. International Federation of Gynaecology and Obstetrics (FIGO) stage distribution was 26%, 46% and 28% for Stages I, II and III, respectively, and 5 year survival was likewise 94% 63% and 37%. Age and histological type or grade were not found to influence survival. Recurrent disease was recorded in 142 patients; the first site was within the pelvis in 25% and as distant metastases in 17%. Following development of pelvic recurrence median survival was 28 weeks. Salvage surgery was performed in 32 patients, of whom five probably obtained survival benefit. Significant late morbidity was seen in a total of 71 patients (19%); in seven patients this was at more than one site. Late morbidity to the small bowel was recorded as Grade 2 in 10 patients and Grade 3 in 13; to the rectum, Grade 2 in 10 patients and Grade 3 in two patients; to the bladder, Grade 2 in 15 patients and to the vagina Grade 2 in 29 patients. Median time to onset for small bowel morbidity was 14 months, for rectum 18 months, for vagina 20 months and for bladder 52 months. 82% of all late morbidity had been seen by 5 years of follow-up, no case of late morbidity of recurrence was seen between 11 and 18 years of follow-up. These results are comparable to those reported for other methods in use at the time the patients were treated.

Concomitant pelvic irradiation, 5-fluorouracil and mitomycin C in the treatment of advanced cervical carcinoma.

This was a Phase II study of 24 late (FIGO) Stage IIb and 39 Stage III patients. External beam radiotherapy was given daily, five days a week, using 15 x 15 cm parallel opposed pelvic fields. The first 20 patients had 45.00 Gy mid-plane dose in 20 fractions, Days 1-28, the last 43 patients had 50.40 Gy in 28 fractions, Days 1-43. This was followed by an intracavitary boost of 17.00 Gy to Point A in two fractions over seven days. The first seven patients had concomitant 5-fluorouracil (5FU) 1 g/m2/day (maximum 1.5 g/day) Days 2-5, 30-33 and 57-60, with mitomycin C 10 mg/m2 (maximum 15 mg) Days 2 and 57. Two patients had WHO Grade 4 cytopenia, and only two were able to have full dose intensity. The 5FU dose was reduced to 0.8 g/m2/day, for Days 2-5 and 30-33; mitomycin C was given on Day 2 only. Treatment morbidity with the reduced chemotherapy intensity was comparable with that of radiotherapy alone. Median follow-up was 16 months (range 6-44). Median survival was 35 months. The results were compared with historical controls treated using the same radiation method alone. Two-year survival for late Stage IIb patients was 67% with the combination and 72% with radiotherapy alone; for Stage III, 67% and 49% respectively. Two-year pelvic control for late Stage IIb was 87% (combination) and 84% (radiotherapy alone) and for Stage III, 61% and 55% respectively. In contrast to reports from other centres, these results do not show an overall significant improvement on radiotherapy alone. A Phase III study may not be practicable.

Hyperthermic isolated perfusion with melphalan in the treatment of advanced malignant melanoma of the lower limb.

A retrospective study of 91 patients with malignant melanoma treated by lower limb isolated hyperthermic regional perfusion was performed. Objective response in patients with evaluable disease was 78 per cent. The role of perfusion as primary treatment of large melanomas of the foot is confirmed and recommended. Control of locoregional disease and limb salvage remains a valuable and attainable therapeutic goal using this technique.

Histologic features predictive of an increased risk of early local recurrence after treatment of breast cancer by local tumor excision and radical radiotherapy.

A histopathologic review was undertaken of 293 patients with breast cancer treated by local tumor excision and radical radiotherapy. In a 6-year period, there were 37 local treatment failures with 16 deaths, and an additional 20 patients died without local recurrence. Pathologic data were available for 272 patients. Multivariate analysis indicated that the pathologic features in an invasive tumor most predictive for local recurrence were the combination of a high proportion of intraduct carcinoma with extensive necrosis (comedocarcinoma). Of 18 patients with these features, nine have had early local recurrence (a risk of 50% with these features vs 10% in those without), and four have died (a risk of 22% against 12%). Despite the short follow-up, the results already appear significant, and the study is ongoing. The importance of comedonecrosis in the intraduct component as a prognostic indicator in invasive carcinoma of the breast is not widely recognized and might constitute a relative contraindication to conservative treatment of the breast.

Pathological features of invasive breast cancer associated with a high risk of local recurrence after tumour excision and radical radiotherapy.

The histological slides of 273 breast cancer patients, treated by local excision and radiotherapy and followed up for 2-6 years, were reviewed in search of factors predictive of local recurrence. Local recurrence was found to be most closely related to the combination of a high proportion of intraduct carcinoma and extensive necrosis (comedocarcinoma). This latter group of 18 patients had a local recurrence rate of 50%. These patients are of particular interest since they form a small group with readily identifiable histological features.

Interim results of treatment of breast cancer with breast conservation for all patients.

Since April 1979, all female patients who have come to this unit with carcinoma of the breast have received primary treatment with breast conservation. Until December 1984, 288 patients underwent local excision of the tumor with radical radiotherapy. After 1 to 5 years, the overall and recurrence-free survival rates have been comparable with those expected after mastectomy. Local recurrence occurred in 7 of 76 (9%) patients who had stage I disease, and in 16 of 181 (9%) and 3 of 27 (11%) patients with stage II and stage III disease, respectively. Eleven patients required a mastectomy to control recurrence of the cancer, and of the 29 patients who died of breast cancer, only three had symptomatic local disease at the time of death.

The functional results following primary treatment of breast cancer with breast conservation.

Forty-seven patients who had local excision ('lumpectomy') and radical radiotherapy for carcinoma of the breast in 1982 were assessed at 1 year for the functional result. The appearance of both breast was identical in 34%, while in 10% serious distortion had occurred. No matchline effect or severe telangiectasia were seen, and no patient had arm oedema, restricted arm movements or severe pain. The results were not significantly better (1) following iridium implantation than external boost, (2) after periareolar rather than other incisions, and (3) in patients with small and medium rather than large breasts.

Alkaline phosphatase for immunocytochemical labelling: problems with endogenous enzyme activity.

Alkaline phosphatase may be used as a label for immunocytochemistry and can be demonstrated in tissue sections using the single step naphthol phosphate method. Endogenous enzyme activity may not be destroyed by fixation in formalin, formol alcohol, Carnoy's or Baker's solutions and should be inhibited before results are assessed. Either Bouin's solution or periodic acid followed by potassium borohydride are satisfactory inhibitor and do not adversely affect immunocytochemical results.