Partially transformed, anchorage-independent human diploid fibroblasts result from overexpression of the c-sis oncogene: mitogenic activity of an apparent monomeric platelet-derived growth factor 2 species.

A human c-sis cDNA in an expression vector was introduced into human diploid fibroblasts by transfection or electroporation. Fibroblast clones showing an aberrant, densely packed colony morphology were isolated and found to overexpress a 3.6-kilobase sis mRNA species and associated immunoprecipitable platelet-derived growth factor (PDGF) 2 proteins. Parallel analyses in cell clones of sis mRNA expression and colony formation in agar indicated that, above a threshold, a linear, positive correlation existed between sis overexpression and acquired anchorage independence. The sis-overexpressing cells formed transient, regressing tumor nodules when injected into nude mice, consistent with the finite life span which they retained. Protein products generated from the transfected c-sis construct in two overexpressing clones were immunoprecipitated with anti-human PDGF antibodies. One clone contained an apparent PDGF dimer of 21 kilodaltons; the second clone contained only an apparent PDGF monomer of 12 kilodaltons, which was shown to account for all of the mitogenic activity present in the cells, essentially all of which was concentrated in the membrane fraction. The results demonstrate a clear link between sis overexpression and acquisition of a partially transformed, anchorage-independent phenotype, and when combined with previous observations of sis overexpression in human tumors, clearly implicate sis overexpression as a genetic mechanism which contributes to human cell transformation.

Mutation at separate gene loci in Salmonella typhimurium TA100 related to DNA nucleotide modification by stereoisomeric benzo(a)pyrene 7,8-diol-9,10-epoxides.

Suspensions of Salmonella typhimurium TA100 or TA1535 cells were exposed to pure enantiomeric forms or racemic mixtures of 3H-labeled benzo(a)pyrene anti- or syn-7,8-dihydrodiol-9,10-epoxide. Diol-epoxide-induced cytotoxicity and mutation frequencies at the hisG and gpt loci were determined. Hydrolysates of diol-epoxide-modified bacterial DNA were also examined by high-performance liquid chromatography and the primary structure and level of diol-epoxide-nucleoside adduct species were related to the observed frequencies of reverse mutations at the mutant hisG46 codon (histidine prototrophy) or forward mutations at the gpt locus (8-azaguanine resistance). Significant differences in mutagenic efficiency (i.e., mutation frequency per mol DNA adduct) were observed for the different enantiomeric diol-epoxides (-anti = +/- syn much greater than, + anti) and the mutagenic efficiencies were the same at both loci. The combined results of the mutation and adduct characterizations suggest that there are basic differences in the structural configuration of each adduct species which are recognized during errant DNA repair and as a result lead to base changes at a frequency which is relatable to the configuration of the original adduct lesion.

Central lymphatic irradiation for stage III nodular malignant lymphoma: long-term results.

PURPOSE: To report the long-term results of central lymphatic irradiation for stage III nodular malignant lymphoma. PATIENTS AND METHODS: Between 1969 and 1985, 34 patients (26 with nodular poorly differentiated lymphoma, four with nodular mixed lymphocytic/histiocytic lymphoma, and four with nodular histiocytic lymphoma) were treated with central lymphatic irradiation. Median age of the group was 51 years (range, 30 to 73). There were 15 men and 19 women. Staging work-up included a physical examination and bone marrow biopsy in all patients. Seventy-four percent had a lymphangiogram (LAG) and 44% a laparotomy (LAP). Eighty-two percent had either a LAP or a LAG. Thirty-two patients were Ann Arbor stage IIIA and two were stage IIIB. All patients received lymphatic irradiation that encompassed cervical, supraclavicular, axillary, mediastinal, paraaortic, mesenteric, pelvic, and femoral lymphatics to total doses ranging from 20 to 30 Gy in 1.0- to 1.8-Gy fractions. Waldeyer's ring was initially treated in 17 patients. Follow-up information is available on all 34 patients. Median follow-up is 9 years, 8 months (range, 15 to 244 months). RESULTS: Life-table actuarial overall, disease-free, and cause-specific survival rates at 15 years are 28%, 40%, and 46%, respectively. Only one relapse was observed after 9 years. Disease-free survival was significantly improved in patients with five or fewer sites of involvement (P = .02). Age, sex, B symptoms, histology, and technique of irradiation were not prognostically significant. Salvage therapy, including further irradiation and/or chemotherapy, was delivered to 20 patients. Ten percent of these patients remain alive without evidence of disease. Toxicity data were available for the patients treated at the Medical College of Wisconsin (MCW). Radiation Therapy Oncology Group (RTOG) acute hematologic, gastrointestinal, and salivary toxicity scores were < or = 2 in 83% of patients. Late toxicity scores were < or = 2 in 96%. Persistent xerostomia was noted in 23% of patients who received initial treatment to Waldeyer's ring. CONCLUSION: These results suggest that initial comprehensive central lymphatic irradiation may be the preferred approach to achieve a durable relapse-free interval for this group of patients.

Ionizing radiation greatly improves gene transfer efficiency in mammalian cells.

The vast majority of clinical protocols involving gene therapy today rely on viral vectors for gene transduction. The primary reason that plasmid vectors have not been widely used for gene therapy trials is their relatively low rate of stable gene transfer. We show here that ionizing radiation can improve plasmid transfection efficiency in both normal and neoplastic human and mouse cells. As high as 1,400-fold improvement in transfection efficiency can be seen in primary human fibroblasts treated with 9 Gy. Radiation improves transfection efficiency in a dose-dependent manner of only linearized plasmid DNA in transformed or immortalized cells, but of both linearized and supercoiled plasmid in normal human fibroblasts. The gene transfer dose-response curves are linear for neoplastic cell lines and exponential for primary cell lines. This suggests that radiation can improve gene integration by at least two mechanisms, one that may require free DNA ends and one that does not. The 2-hr delay described here, from the time of irradiation to the beginning of enhanced gene integration, suggests an inducible process that becomes active after the bulk of the radiation damage has been repaired. Our data further suggest that radiation may be useful to target human gene therapy using plasmid vectors.

High-efficiency stable gene transfer of adenovirus into mammalian cells using ionizing radiation.

We report a novel method for targeting adenovirus-mediated gene delivery. By irradiating mammalian cells prior to adenoviral transduction, adenoviral gene transfer is greatly improved and the adenoviral genome integrates into cellular DNA. In this work, human and rodent cell lines were irradiated and subsequently transduced with the adenovirus vector Ad5CMVlacZ. Initial levels of transduction were as much as 40-fold higher in irradiated cells, and this improvement in transduction was radiation dose dependent. The duration of lacZ expression in irradiated cells was also much longer than in nonirradiated cells and reached a plateau after 21 days. At doses of 7 Gy, long-term (< 50 day) expression of lacZ could be detected in 15% of cells by flow cytometry. This long-lasting expression of lacZ was due to viral DNA integration into the host genome. Thus, pretreatment of cells with ionizing radiation improves both immediate transduction efficiency and duration of transgene expression. This may lead to the development of new protocols combining radiation and gene therapy in treating human malignancy.

The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide.

Instability of hydrogen peroxide solutions was noted during the experimental exposure of human cells in culture to hydrogen peroxide in experiments designed to study the production and repair of DNA single-strand breaks. A hydrogen peroxide concentrate was diluted into culture medium, which was then added to experimental cell cultures at various times, with all cultures assessed for DNA damage at 2 h. Only cells treated by the first addition had observable DNA damage. This result was unexpected since these cells had had the maximum repair time. It was determined that the hydrogen peroxide had been eliminated by the culture medium. To determine the mechanism of this elimination, 200 microM hydrogen peroxide was added to various cell culture components, and the solutions were assayed for hydrogen peroxide after 1 h at 37 degrees C. Although most components (except the balanced salts) showed some hydrogen peroxide degradation, it was found that sodium pyruvate was most effective, by a wide margin, in eliminating hydrogen peroxide and its toxic effects. This was confirmed by addition of pyruvate to balanced salt solutions or buffers, and observing the same elimination of hydrogen peroxide. We subsequently found a few earlier reports describing the decarboxylation reaction between hydrogen peroxide and pyruvate, but no kinetic measurements have been published and there seems to be no general appreciation for the very high efficiency of this reaction. The present work presents a preliminary assessment of the importance of pyruvate in the study of hydrogen peroxide and other reactive oxygen species in mammalian cell culture.

Randomized phase III study of chemoradiation with or without amifostine for patients with favorable performance status inoperable stage II-III non-small cell lung cancer: preliminary results.

A prospective randomized study was conducted to determine whether amifostine (Ethyol) reduces the rate of severe esophagitis and hematologic and pulmonary toxicity associated with chemoradiation or improves control of non-small cell lung cancer (NSCLC). Sixty patients with inoperable stage II or III NSCLC were treated with concurrent chemoradiotherapy. Both groups received thoracic radiation therapy (TRT) with 1.2 Gy/fraction, 2 fraction per day, 5 days per week for a total dose 69.6 Gy. All patients received oral etoposide (VP-16), 50 mg Bid, 30 minutes before TRT beginning day 1 for 10 days, repeated on day 29, and cisplatin 50 mg/m(2) intravenously on days 1, 8, 29, and 36. Patients in the study group received amifostine, 500 mg intravenously, twice weekly before chemoradiation (arm 1); patients in the control group received chemoradiation without amifostine (arm 2). Patient and tumor characteristics were distributed equally in both groups. Of the 60 patients enrolled, 53 were evaluable (27 in arm 1, 26 in arm 2) with a median follow-up of 6 months. Median survival times were 26 months for arm 1 and 15 months for arm 2, not statistically significantly different. Morphine intake to reduce severe esophagitis was significantly lower in arm 1 (2 of 27, 7.4%) than arm 2 (8 of 26, 31%; P =.03). Acute pneumonitis was significantly lower in arm 1 (1 of 27, 3.7%) than in arm 2 (6 of 26, 23%; P =.037). Hypotension (20 mm Hg decrease from baseline blood pressure) was significantly more frequent in arm 1 (19 of 27, 70%) than arm 2 (1 of 26, 3.8%; P =.0001). Only 1 patient discontinued treatment because of hypotension. These preliminary results showed that amifostine significantly reduced acute severe esophagitis and pneumonitis. Further observation is required to assess long-term efficacy.

Treatment planning using a dose-volume feasibility search algorithm.

PURPOSE: An approach to treatment plan optimization is presented that inputs dose--volume constraints and utilizes a feasibility search algorithm that seeks a set of beam weights so that the calculated dose distributions satisfy the dose--volume constraints. In contrast to a search for the "best" plan, this approach can quickly determine feasibility and point out the most restrictive of the predetermined constraints. METHODS AND MATERIALS: The cyclic subgradient projection (CSP) algorithm was modified to incorporate dose--volume constraints in a treatment plan optimization schema. The algorithm was applied to determine beam weights for several representative three-dimensional treatment plans. RESULTS: Using the modified CSP algorithm, we found that either a feasible solution to the dose--volume constraint problem was found or the program determined, after a predetermined set of iterations was performed, that no feasible solution existed for the particular set of dose--volume constraints. If no feasible solution existed, we relaxed several of the dose--volume constraints and were able to achieve a feasible solution. CONCLUSION: Feasibility search algorithms can be used in radiation treatment planning to generate a treatment plan that meets the dose--volume constraints established by the radiation oncologist. In the absence of a feasible solution, these algorithms can provide information to the radiation oncologist as to how the dose--volume constraints may be modified to achieve a feasible solution.

Respiratory-driven lung tumor motion is independent of tumor size, tumor location, and pulmonary function.

PURPOSE: To determine whether superior-inferior lung tumor motion is predictable by tumor size or location, or pulmonary function test results. METHODS AND MATERIALS: Superior-inferior tumor motion was measured on orthogonal radiographs taken during simulation of 22 patients with inoperable lung cancer diagnosed by orthogonal radiographs. RESULTS: The tumor size averaged 5.5 +/- 3.1 cm (range 1.5-12 cm). Seven of 11 central tumors demonstrated some motion compared with 5 of 11 peripheral tumors. Four of 5 upper lobe tumors moved compared with 8 of 17 tumors that were either middle or lower lobe lesions. The mean fourth rib motion was 7.3 +/- 3.2 mm (range 2-15). The mean FeV(1) was 1.8 +/- 1.2 (range 0.55-5.33. The mean diffusing capacity of the lung for carbon monoxide was 14.0 +/- 6.5 (range 7.8-21.9). The mean total lung capacity was 6.5 +/- 1.2 (range 3.3-8.4). None of these parameters correlated with tumor motion. Although lateral tumor motion could not be consistently determined, 1 tumor moved 10 mm anterior-posteriorly. CONCLUSIONS: Lung tumors often move significantly during respiration. Tumor motion is not predictable by tumor size or location, or pulmonary function test results. Therefore, tumor motion must be measured in all patients. Measurement in three dimensions will likely be necessary to maximize the irradiated lung volumes or choose beam arrangements parallel to the major axis of motion.

Interaction of midazolam and morphine in the spinal cord of the rat.

The antinociceptive properties, as measured by the tail-flick and hot-plate tests, and the motor effects of an intrathecally-administered benzodiazepine agonist midazolam, alone, and in combination with morphine, was examined in rats. Midazolam alone produced a weak but dose-dependent (20-60 micrograms) antinociceptive effect in addition to a clear motor dysfunction at larger doses (60-100 micrograms). An inactive dose of intrathecally-administered midazolam (20 micrograms) produced a leftward shift in the dose-response curve for intrathecally administered morphine, in the thermal antinociceptive tests. This supra-additive effect was antagonized by naloxone (1 mg/kg). The data suggest a synergistic interaction between mu- and GABAA-receptors in the spinal processing of thermally-evoked pain.

Novel approaches to locally advanced unresectable non-small cell lung cancer.

The management of advanced non-small cell lung cancer (NSCLC) is rapidly evolving. Advances in combined chemo-radiation therapy have led to improvements in patient survival which are statistically significant, but most patients still succumb to their disease. New chemotherapeutic agents, such as taxanes (paclitaxel, docetaxel), topoisomerase inhibitors (topotecan, irinotecan), and novel analogs (gemcitabine, vinorelbine), may offer the promise of improved outcome, but have not yet been tested in phase III trials. Molecular therapeutics, such as gene therapy, drugs that target specific oncogene activation (such as Ki-ras inactivation by farnesyl transferase inhibitors), and hypoxic cell toxins (such as tirapazamine), are in clinical trials. The optimum use of these agents awaits more rapid and widespread molecular diagnostics. Finally, technological advances in radiotherapy will allow higher tumor doses, while minimizing doses to dose-limiting normal structures, such as the esophagus, normal lung and heart. We describe a move towards molecular strategies, both for therapy and diagnostics, that may result in more effective treatment. While the outcome for patients with advanced non-small cell lung carcinoma is still poor, new agents are being developed rapidly and offer the hope of improved survival.

Benzo[a]pyrene-diol-epoxide-induced anchorage-independence in diploid human fibroblasts. Analysis of cellular protooncogenes.

Treatment of diploid human fibroblasts with stereoisomeric benzo[alpha]pyrene anti and syn diol epoxides has been shown to induce anchorage-independent clones of cells with a dose dependence and frequency [(0.5-12) X 10(-4)] not significantly different from mutations at the hypoxanthine-guanine phosphoribosyltransferase locus [(1-8) X 10(-4)] in these cells. The majority of the anchorage-independent clones that were picked retained their mutagen-induced, anchorage-independent phenotype through at least 20 generations of expansion in monolayer culture. No variant cells showing extended life-span were detected among survivors in any of the mutagen treatment groups (less than 1.6 X 10(-7) frequency). Extensive analysis of a pool of 15 cellular protooncogenes (Ha-ras, Ki-ras, N-ras, mos, fos, fes, myc, abl, sis, myb, erbA, erbB, src, raf, N-myc), using Southern and northern blot analysis, was done to determine whether mutagen-induced rearrangement, amplification or overexpression of any of these genes was responsible for the mutagen-induced, anchorage-independent phenotype. We found no evidence that the genomic arrangement or expression level of any of these genes had been altered, thus indicating that an alternative form of mutation, or an alternative gene not included in this screening was responsible for the mutagen-induced, anchorage-independent phenotype.

Radiation-induced recombination is dependent on Ku80.

We have recently shown that irradiating cells prior to transfection induces recombination, as manifested by increased stable transduction of both plasmid and adenoviral vectors. We hypothesized that Ku proteins, which have previously been shown to be involved in both recombination and the repair of DNA damage after irradiation, would likely be important mediators of radiation-induced recombination. The present work demonstrates that Ku80 is essential for radiation-induced recombination. While human and hamster Ku80 are equally effective at restoring the transfection efficiency and radiation resistance of xrs-5 cells, human Ku80 is much more effective at radiation-induced recombination than hamster Ku80. This difference is not due to differences in Ku80 expression or DNA end-binding activity, but it may be due to structural differences between human and hamster Ku80.

The significance of atypical cervical cytology in an older population.

To ascertain the significance of squamous atypia encountered during routine Papanicolaou smear screening in an older population, we reviewed 115 consecutive patients over age 50 seen during a 3-year period. Evaluation included repeating the smear and performing colposcopy in all patients. Colposcopically directed biopsy and endocervical curettage were performed when appropriate. Sixty-seven patients (58.3%) had atrophy, 43 (37.4%) were normal, two (1.7%) had cervical intraepithelial neoplasia grade 1, two (1.7%) had cervical intraepithelial neoplasia grade 2, and one (0.9%) had human papillomavirus (HPV) infection. There was a strong association between squamous atypia and estrogen deficiency. With the correction of the estrogen deficiency, the squamous atypia reverted to normal in a statistically significant percentage of patients. A Papanicolaou smear report of squamous atypia in women over age 50 should not be considered normal; further evaluation is required. The incidence of cervical or vaginal intraepithelial neoplasia and HPV infection is much lower than reported in studies involving younger patients.

Primary invasive carcinoma of the vagina.

A retrospective study of 29 patients with invasive carcinoma of the vagina was completed at The Milton S. Hershey Medical Center, Pennsylvania State University, for a ten-year period from 1976-1986. The overall incidence was 1.3% of all gynecologic malignancies. Twenty-four patients (83%) had squamous cell carcinoma and five (17%) had adenocarcinoma. Squamous cell carcinoma was most commonly located in the upper anterior and lateral vaginal vaults, whereas adenocarcinoma was found more often in the lower anterior and lateral vaginal vaults. The majority of the patients (96%) were managed by a combination of whole-pelvis irradiation and brachytherapy. Twenty-four percent of the patients had a recurrence in the vagina only, indicating the need for better local control. The overall survival rate was 48%. Patients with previous hysterectomy were more likely to develop serious treatment-related complications.

Chronic antagonist infusion does not increase morphine antinociception in rat spinal cord.

Chronic spinal infusion of the opiate antagonists naloxone or naltrexone fail to influence the antinociceptive effect of subsequent intrathecal morphine on the hot plate test in rats compared to saline-infused controls. These results contrast the functional supersensitivity to morphine seen after long-term systemic opiate antagonist administration and support the hypothesis that dopaminergic interactions, lacking in the spinal cord, are necessary for antagonist-induced opioid receptor upregulation.

Endogenous opioid system down-regulation during hibernation in amphibians.

In late summer, Northern grass frogs, Rana pipiens, intraspinally administered dynorphin produces a potent, dose-dependent antinociceptive action as measured by the acetic acid test used to evoke a hindlimb wiping response. Surprisingly, in fall, frogs which have entered hibernation, intraspinal dynorphin produces no antinociception action. Intraspinal morphine shows a decreased effect in fall frogs while systemic morphine is equi-effective in summer and fall frogs. Immobilization stress, previously shown to be mediated by endogenous opioid systems in this amphibian, produces a robust increase in nociceptive thresholds in summer frogs while the nociceptive thresholds of fall frogs are unaffected by this procedure. Summer frogs adapted to cold room (4 degrees C) show a significant decrease in nociceptive thresholds compared to cohorts kept at room temperature, and cold-adapted frogs returned to room temperature show a naloxone-attenuated increase in nociceptive threshold. Collectively, these data suggest that endogenous opioid systems in these northern frogs are down-regulated during fall hibernation.

Naloxone blocks the analgesic action of levorphanol but not of dextrorphan in the leopard frog.

Intraspinal injection of levorphanol (3 micrograms) at the lumbar area of the leopard frog, Rana pipiens, induced analgesia which was completely blocked by co-injection of naloxone (3 micrograms), whereas dextrorphan (3 micrograms) induced analgesia which was unaffected by naloxone. Subcutaneous levorphanol (20 or 80 mg/kg) induced a dose-dependent analgesia which was blocked by concurrent naloxone (2 mg/kg), while only the higher dose of dextrorphan (80 mg/kg) induced analgesia which was unaffected by concurrent naloxone at 8 or 80 mg/kg. These data are the first to indicate naloxone-insensitive, dextrorphan-induced analgesia.

Spinal action of dermorphin, an extremely potent opioid peptide from frog skin.

Studies on the characteristics of spinally administered dermorphin, a novel heptapeptide isolated from the skin of South American Phyllomedusa frogs, indicated that this agent is 3-5000X more active spinal morphine on the hot plate, tail flick and writhing tests. This agent displays naloxone reversibility and cross tolerance to spinal morphine, and possesses all of the characteristics of a mu opiate receptor agonist.

Changes of opioid binding density in the rat spinal cord following unilateral dorsal rhizotomy.

Mu, delta and kappa opioid receptors in the vertebrate spinal cord mediate the potent antinociceptive effects of opioid agonists administered onto the spinal cord. The present experiments were conducted to determine the effect of unilateral dorsal rhizotomy on mu, delta and kappa spinal opioid binding sites. Measurements of opioid binding were made at 1, 2, 4 or 8 days after rhizotomy and comparisons were made to intact animals. The changes in mu, delta and kappa opioid binding sites were determined by receptor autoradiography using the highly selective radioligands [3H]sufentanil, [3H]DPDPE and [3H]U69593, respectively. Within autoradiograms of each spinal cord, three regions on each side of the spinal cord were targeted for densitometric analysis: laminae I-II (medial), V (lateral) and X. When effects of unilateral rhizotomy within animals were assessed by comparison of the density of binding on the side ipsilateral to the rhizotomy to the contralateral side, decreases in the binding of all three radioligands were observed in laminae I-II on the side of the spinal cord ipsilateral to the rhizotomy at 2-8 days postlesion. A significant reduction in binding was also noted for mu and delta sites in lamina V after 8 days and for delta binding in lamina X at 2 and 4 days on the side ipsilateral to the rhizotomy. However, when densities of binding sites were compared with the corresponding regions in control, it was clear that dorsal rhizotomy resulted in significant changes in opioid binding on both sides of the spinal cord; changes differed for each type of opioid binding site. On the contralateral side of the spinal cord, rhizotomy caused a significant decrease of mu opioid sites 1 day after the lesion and showed partial recovery by day 8. Delta opioid sites were also significantly decreased as early as 1 day postlesion, but did not recover. Kappa opioid sites did not change at 1 day after the rhizotomy but increased on day 2, decreased on day 4 and fully recovered 8 days after rhizotomy. The present results support the hypothesis that a significant proportion of spinal mu, delta and kappa opioid binding sites are present on the central terminations of primary afferents. Finally the present data are the first to report a contralateral effect of the unilateral rhizotomy on spinal opioid binding sites. The contralateral changes in binding were specific to the type of opioid site examined, time after the surgery and region of the spinal cord examined.