Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing.

We analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs there is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

Prophylactic and therapeutic actions of supplemental beta-carotene in mice inoculated with C3HBA adenocarcinoma cells: lack of therapeutic action of supplemental ascorbic acid.

Decreased tumor incidence, increased latent period, and increased survival time were observed in C3H/HeJ mice fed supplemental beta-carotene for 3 days and then inoculated with 10(4) C3HBA (syngeneic) tumor cells. In addition, C3H/HeJ, C3H/He, and CBA/J mice, fed supplemental beta-carotene beginning immediately after they were inoculated with 2 X 10(5) C3HBA tumor cells, showed decreased tumor growth and increased survival time. When beta-carotene was fed to mice in which palpable tumors were already present, it similarly slowed tumor growth and extended animal survival time. Ascorbic acid supplementation (5 g/kg diet), introduced into the experiment as a possible synergist for beta-carotene's antitumor action, was without therapeutic action when tested in the presence or absence of beta-carotene supplements. The basal diet, a standard commercial mouse chow, contains more vitamin A than the National Research Council's recommended dietary allowance for normal rodents and supports normal growth, reproduction, and longevity of normal mice. The work reported here is the first demonstration of the antitumor action of beta-carotene in animals with a transplanted tumor.

Moloney murine sarcoma virus tumors in CBA/J mice: chemopreventive and chemotherapeutic actions of supplemental beta-carotene.

Decreased tumor frequency, increased latent period, and increased rate of tumor regression were observed in male inbred CBA/J mice fed supplemental beta-carotene before and/or after they were inoculated with the Moloney sarcoma virus. When beta-carotene feeding was begun after tumors were already present, it markedly increased the rate of tumor regression. beta-Carotene minimized the virus-induced thymus gland involution that accompanies tumor growth, and this action on the thymus gland was believed to underlie part of beta-carotene's antitumor activity. The basal diet, a standard commercial mouse chow containing more vitamin A than the National Research Council recommends as a daily allowance for rodents, supported normal growth, reproduction, and longevity of normal mice. The work reported here is the first demonstration of the antitumor action of beta-carotene in mice inoculated with an oncogenic virus.

Granulopoiesis associated with the C3HBA tumor in mice.

Female C3H/HeJ mice were inoculated with syngeneic breast adenocarcinoma cells (C3HBA). A progressive neutrophilia developed as the tumors grew. A linear relationship was demonstrated between the tumor diameter and the extent of the neutrophilia. Local tumor excision caused a rapid fall (3 days) in the neutrophil count. Media conditioned with tumor cells, normal mouse kidney, and bone marrow of normal or tumor-bearing mice were prepared. Tumor cell-conditioned medium was found to have marked stimulating activity for granulocytic colony formation of mouse bone marrow cells. Sera from tumor-bearing mice also had colony-stimulating activity. This finding strongly suggested that the neutrophilia was caused by the release of a neutrophil-stimulating factor from the tumor.

Antipyretic and antiviral action of vitamin A in Moloney sarcoma virus- and poxvirus-inoculated mice.

Six week-old male CBA/J mice fed a commercial powdered laboratory chow or the same chow supplemented with vitamin A palmitate (150,000 U/kg) were inoculated with either the Moloney strain of murine sarcoma virus (M-MuSV) or poxvirus. Central body temperature was measured daily. Both viruses elicited fevers, but the fevers were less pronounced and of shorter duration in the mice ingesting the vitamin A-supplemented diet. Palpable M-MuSV-induced tumors appeared later, were less frequent, grew more slowly, and were resorbed sooner in the mice fed the vitamin A supplement. Similarly, in these mice the appearance of pox lesions was delayed, their numbers reduced, and their disappearance hastened.

Morbidity and mortality reduction by supplemental vitamin A or beta-carotene in CBA mice given total-body gamma-radiation.

Male CBA mice received graded doses (450-750 rad) of total-body gamma-radiation (TBR) from a dual-beam 137Cs irradiator. Commencing directly after TBR, 2 days later, or 6 days later, groups of mice received supplemental vitamin A (Vit A) or beta-carotene (beta-Car), compounds previously found to reduce radiation disease in mice subjected to partial-body X-irradiation. Given directly after TBR, supplemental Vit A decreased mortality, evidenced by increases in the radiation dose required to kill 50% of the mice within 30 days (LD50/30). In one experiment, Vit A increased the LD50/30 from 555 to 620 rad; in another experiment, Vit A increased the dose from 505 to 630 rad. Similarly, in a third experiment, supplemental beta-Car increased the LD50/30 from 510 to 645 rad. Additionally, each compound increased the survival times, even of those mice that died within 30 days. In addition to reduction of mortality and prolongation of survival time, supplemental Vit A moderated weight loss, adrenal gland hyperemia, thymus involution, and lymphopenia--all signs of radiation toxicity. Delaying the supplementation for 2 days after irradiation did not greatly reduce the efficacy of Vit A; however, delaying supplementation for 6 days decreased its effect almost completely.

Antitumor action of vitamin A in mice inoculated with adenocarcinoma cells.

Vitamin A palmitate was incorporated into a laboratory chow (150,000 IU/kg diet) and fed ad libitum to C3H/HeJ female mice inoculated with 1 times 10-6 C3HBA tumor cells, beginning the day of inoculation. Control female mice of the same strain similarly inoculated were fed the laboratory chow alone. Vitamin A did not affect rate for the first 19 days, after which growth rates were independent of treatment. Vitamin A-treated mice survived for significantly longer times than did control mice.

Regression of C3HBA mouse tumor due to X-ray therapy combined with supplemental beta-carotene or vitamin A.

Male CBA/J mice, ingesting a vitamin A- and beta-carotene-sufficient laboratory chow, were inoculated in a hind limb with 2 X 10(5) C3HBA adenocarcinoma cells. When the mean tumor size was 6.2 mm, the mice were divided randomly into groups; some groups received supplemental vitamin A or beta-carotene, some received 3,000 rad local radiation to the tumor, and others received both radiation and one of the supplements. All mice that received only radiation or one of the dietary supplements died within 3 months. When local irradiation and supplemental vitamin A or beta-carotene were coupled, "complete" tumor regression occurred in every case (12/12), and tumor regrowth in and death of the mice occurred in only 1 of 12 in each of these groups during the succeeding 12 months. One year after irradiation and dietary supplementation, half the surviving mice were switched back to the control chow. During the next year, none of the mice remaining on the vitamin A or beta-carotene supplements developed tumors; however, of 6 mice switched from vitamin A, 5 had tumors that reappeared. In contrast, tumors recurred in only 2 of 6 mice after they were switched from beta-carotene. A second experiment yielded similar results. These results show that both vitamin A and beta-carotene supplementation added remarkably to the antitumor effect of local irradiation. beta-Carotene supplementation produced a greater residual antitumor action than vitamin A supplementation after the supplements were discontinued, which may have been due to greater tissue storage of beta-carotene.

Comparison of amplified and unamplified c-myc gene structure and expression in human small cell lung carcinoma cell lines.

A human small cell lung cancer cell line (H82) demonstrates 40- to 50-fold amplification of the c-myc gene but expresses at least 250-fold more steady-state c-myc messenger RNA than an unamplified small cell lung cancer cell line (H378) with no detectable expression of c-myc. We compared the chromatin structure of c-myc in H82 to that in H378 using DNase I sensitivity and DNA methylation patterns. DNase I hypersensitivity sites were identical in H82 and H378 and were similar to the pattern seen in a B-lymphoblastoid cell line, despite extensive amplification of c-myc in H82. Methylation patterns were also very similar in H82 and H378, with hypomethylation or partial methylation at the c-myc coding regions and the flanking 5' sequences, despite the absence of detectable c-myc expression in H378. Therefore, the predominant chromatin structural patterns do not appear to correlate with observed differences in gene expression. In addition, these studies demonstrate that the patterns of DNase I hypersensitivity and of methylation can remain intact during a 40- 50-fold gene amplification, as observed for the c-myc gene in H82.

Intravascular lymphomatosis: a clinicopathologic study of 10 cases and assessment of response to chemotherapy.

PURPOSE: We report a clinicopathologic study of 10 cases of intravascular lymphomatosis (IVL) seen at a single institution, and assess the response to chemotherapy in these patients, as well as those collected from a literature review. PATIENTS AND METHODS: The clinical, pathologic, and immunophenotypic features of 10 cases of IVL diagnosed at the Johns Hopkins Hospital since 1977 were studied. Follow-up information was obtained in each case by consultation with the treating physician. In addition, cases of IVL reported previously in which patients were treated with chemotherapy and for which follow-up data were available at the time of publication were reviewed. RESULTS: In the present series of 10 cases, the most common clinical features were fever of unknown origin (FUO), mental status changes, and rash. Diagnostic specimens were obtained from a variety of sources, including brain, skin, prostate, liver, kidney, and gallbladder. All of the four patients treated with combination chemotherapy are alive and two have achieved long-term survival (48 and 45 months, respectively); the remaining two are alive and in complete remission (CR) after short follow-up duration of 6 months. Among 35 patients reported in the literature who received chemotherapy (including four from this series), 43% attained a CR and were free of disease at the time of publication. None of the three patients in our series who received localized therapy (surgery with or without radiation therapy) is alive (mean survival duration, 9 months). For the three patients diagnosed at postmortem examination, the mean interval between onset of symptoms and death was 3 months, and disease was widespread. CONCLUSION: These findings suggest that IVL represents a high-grade non-Hodgkin's lymphoma (NHL) with a propensity for systemic dissemination, and that CR and long-term survival may result in patients treated with aggressive combination chemotherapy.

A trial of combination chemotherapy followed by hormonal therapy for previously untreated metastatic carcinoma of the prostate.

We administered combination chemotherapy with cyclophosphamide, doxorubicin, and cisplatin to 25 previously untreated patients with metastatic prostate cancer in order to assess the efficacy of chemotherapy before any hormonal manipulation. Hormonal therapy was administered only after progression of disease to chemotherapy. All 25 patients were followed until time of death and all were able to receive hormonal therapy. We did not find substantially improved response rates when combination chemotherapy was applied before endocrine treatment since the 33% objective response rate to chemotherapy was only minimally higher than the response in our patients who had failed hormonal therapy and then received identical or similar chemotherapy. Furthermore, the introduction of intensive combination chemotherapy before hormonal therapy in our study did not result in any striking improvement in overall survival compared with patients who received initial hormonal therapy in many other studies. Responses to chemotherapy were not attributable to suppression of serum testosterone since all 12 patients with partial response (PR) or stable disease (SD) and four of seven patients with no response (NR) had normal testosterone levels at the time of response assessment. The initial use of chemotherapy did not adversely affect the expected high percentage of objective responses (68%) to subsequent hormonal manipulation. The frequency, duration, and quality of responses to hormonal therapy exceeded the responses to chemotherapy. The disappointing responses to chemotherapy reflect the very modest efficacy of even aggressively delivered cytotoxic agents.

Pulmonary toxicity with combined modality therapy for limited stage small-cell lung cancer.

To assess the pulmonary toxicity of radiation therapy combined with chemotherapy v chemotherapy alone, we reviewed the clinical course of 80 patients with limited stage small-cell lung cancer treated in a randomized prospective trial. Life-threatening pulmonary toxicity, defined as bilateral pulmonary infiltrates extending beyond radiation ports with symptoms requiring hospital admission, developed in 11 patients (28%) receiving combined modality therapy and in two (5%) receiving chemotherapy alone. Eight of these 13 patients died from pulmonary complications with no clinical evidence of tumor in five. Pulmonary toxicity initially presented at a median of 63 days (range, 21 to 150 days) after the start of combined modality therapy and at a median of 217 days after chemotherapy alone. Biopsies obtained in 11 patients with severe toxicity revealed only interstitial fibrosis with no evidence of an infectious agent. Review of pretreatment parameters such as age, performance status, and radiation portal area failed to reveal any significant differences between patients with or without pulmonary complications. However, initial pulmonary function tests (PFTs) revealed a significantly lower vital capacity (P = .03) and forced expiratory volume (FEV/1.0 second) (P = .04) in patients with subsequent pulmonary complications. Pulmonary toxicity was significantly more common with combined modality therapy than with chemotherapy alone (P = .017) and worse than expected with radiotherapy alone. Six- or 12-month PFTs in completely responding patients revealed improvement within the chemotherapy alone group and no clear trend within the combined modality group. For the group treated with radiation therapy and chemotherapy, there was significantly less improvement after 6 or 12 months in the forced vital capacity (P less than .005) and FEV/1.0 second (P less than .005) than observed for the group treated with chemotherapy alone. Despite the increased incidence of pulmonary toxicity, overall survival favored the combined modality arm (P = .07). Enhanced local control and disease-free survival appeared to compensate for the initial increased pulmonary morbidity and mortality in the group with combined modality therapy.

High-dose cytotoxic therapy and bone marrow transplantation for relapsed Hodgkin's disease.

Patients with Hodgkin's disease who have failed two or more chemotherapy regimens or who have relapsed after an initial chemotherapy-induced remission of less than 12 months are seldom cured with conventional salvage therapies. We studied the effect of high-dose cytoreductive therapy followed by bone marrow transplantation in 50 such patients with relapsed Hodgkin's disease. Twenty-one patients with histocompatibility locus antigen (HLA)-matched donors had allogeneic marrow transplants, one patient received marrow from an identical twin, and 28 patients without a matched donor received autologous grafts purged with 4-hydroperoxycyclophosphamide. Busulfan plus cyclophosphamide was the preparative regimen for the 25 patients who had received extensive prior irradiation, and the other 25 patients received cyclophosphamide plus total body irradiation. The overall actuarial probability of event-free survival at 3 years was 30%, with a median follow-up of 26 months. The event-free survival following transplantation was influenced by the number of chemotherapy failures and the patient's response to conventional salvage therapy prior to transplant. The 16 patients who were transplanted at first relapse, while still responsive to standard therapy, had a 64% actuarial probability of event-free survival at 3 years. Age, presence of extranodal disease, preparative regimen, and type of graft (autologous v allogeneic) were not significant prognostic factors. The majority of transplant-related deaths were from interstitial pneumonitis; inadequate pulmonary function, multiple prior chemotherapy regimens, and prior chest irradiation all appeared to increase the transplant-related mortality. These results suggest a role for marrow transplantation in a subset of patients with relapsed Hodgkin's disease who are unlikely to be otherwise cured but are still responsive to conventional-dose cytoreductive therapy.

Long-term results of blood and marrow transplantation for Hodgkin's lymphoma.

PURPOSE: To evaluate the long-term outcome after allogeneic (allo) and autologous (auto) blood or marrow transplantation (BMT) in patients with relapsed or refractory Hodgkin's lymphoma (HL). PATIENTS AND METHODS: We analyzed the outcome of 157 consecutive patients with relapsed or refractory HL, who underwent BMT between March 1985 and April 1998. Patients

Wound healing and thymotropic effects of arginine: a pituitary mechanism of action.

Supplemental dietary arginine HCl (ARG-HCl) minimizes immediate post-wounding weight loss, accelerates wound healing, and is thymotropic for uninjured and wounded rats. The present experiments were to determine if arginine-pituitary interactions underlie these effects because arginine is a growth hormone secretagogue. Effects of 1% dietary ARG-HCl supplements (0.5% added to a regular commercial rat diet containing 1.8% ARG, 0.5% in drinking water) were studied in (a) hypophysectomized (hypox) rats supplemented with ACTH, L-thyroxine, testosterone propionate, (b) such hypox rats additionally supplemented with bovine growth (hypox + bGH) hormone, (c) intact rats (Int), and (d) intact rats supplemented with growth hormone (Int. bGH). Group (a) hypox rats healed their wounds as rapidly as intact rats (dorsal skin incision breaking strength, accumulation of reparative collagen in sc polyvinyl alcohol sponges). Group (b) hypox, bGH rats showed increased wound breaking strength and accumulation of reparative collagen in the sc sponges to levels significantly greater than those of intact controls; bGH given to intact controls did not affect these indices of wound healing. Supplemental ARG-HCl given intact rats significantly minimized immediate postoperative weight loss, increased wound breaking strength and sponge reparative collagen accumulation, and increased thymic weight. None of these effects of supplemental ARG-HCl were observed in group (a) hypox rats or group (b) hypox + bGH rats. We conclude that an intact hypothalamic-pituitary axis is necessary for these beneficial effects of supplemental ARG-HCl given wounded rats.

An outlier theory of cancer curability. Tumor cell differentiation as a therapeutic goal.

Combinations of cytotoxic agents that cure a substantial percentage of patients with several childhood and adult malignancies are much less efficacious for the majority of solid tumors. Standard approaches for curability that rely solely on the concept of cytotoxicity may not be applicable for most epithelial and mesenchymal solid malignancies. Differentiation may play a more important role in cancer cure than heretofore suspected. Clinical and experimental evidence supports further investigation into models of inducing tumor cell differentiation. The question of why such models could predict for curative modalities of treatment is discussed.

Abnormal coagulation results in patients with Hodgkin's disease.

Case records of 177 patients admitted with Hodgkin's disease were reviewed to assess the frequency and significance of coagulation abnormalities. Prolongation of the prothrombin time, activated partial thromboplastin time, or thrombin time occurred in 56 patients, 32 percent of all evaluable cases. The most frequent clotting abnormalities involved the prothrombin time, which was increased in 43 patients (24 percent). Prothrombin time prolongation correlated with bulky or advanced disease as defined by stage (p = 0.001), constitutional symptoms (p less than 0.0001), massive mediastinal involvement (p = 0.02), and elevated alkaline phosphatase levels (p less than 0.0001). Abnormal coagulation test results followed the course of disease, normalizing with tumor regression and reappearing during relapse. Despite the surprising incidence of abnormal coagulation results, bleeding complications were reported in only two cases. Patients undergoing invasive procedures in the presence of clotting abnormalities fared no worse than those in whom procedures were cancelled. There is no evidence that complete staging evaluation should be comprised because of these abnormal test values. Extensive hematologic testing revealed no single mechanism to explain the coagulation factor disorders found in Hodgkin's disease.

Vitamin A and retinoic acid: induced fibroblast differentiation in vitro.

The role of vitamin A in wound healing and fibroplasia has been studied extensively in vivo but the mechanism(s) of its action has not been established. In this study the effect of vitamin A and retinoic acid on fibroblast growth and collagen accumulation in vitro was examined. Vitamin A and retinoic acid added to Balb 3T3 mouse fibroblasts in tissue culture resulted in induction of cell differentiation as manifested by a decrease in cell growth rate, enhanced collagen accumulation, and morphologic differentiation. The results of this in vitro study suggest that the stimulatory in vivo effect of vitamin A and retinoic acid on collagen accumulation and fibroplasia in healing wounds is due in a major way to fibroblast differentiation and enhanced collagen synthesis.

Arginine: an essential amino acid for injured rats.

The influence of arginine supplements on growth and healing of skin incisional wounds was studied in rats fed either a chemically defined diet lacking arginine or a laboratory chow containing 1.8% arginine. Rats fed the arginine-free diet grew more poorly than did arginine-supplemented rats (1.8 vs. 7.0 gm/day) in the preoperative period. After operation arginine-deficient animals grew very poorly (1 gm/day), while arginine-supplemented rats gained 4.3 gm/day. Arginine-deficient animals showed impaired wound healing, as judged by the breaking strengths of their incisions 10 days after wounding (228 vs. 293 gm for the arginine-supplemented rats). Arginine-deficient rats also showed decreased collagen deposition in a specific wound site, as indicated by the decreased content in hydroxyproline in sponge granulomas (2.5 vs. 4.2 mg/100 mg. of sponge for the arginine-supplemented rats). In rats fed commercial chow, 1% arginine decreased the postoperative weight loss associated with injury (0.7 vs. 5.2 gm) in one experiment and improved wound strength in two experiments (312 vs. 188 gm in one experiment and 309 vs. 246 gm in another). Arginine also increased hydroxyproline deposition in a specific wound area (5.5 vs. 4.1 mg in one experiment and 3.1 vs. 1.9 mg. in another). It is concluded that arginine has two roles in wounded animals. It is essential for the synthesis of the increased amounts of reparative collagen required for wound healing, and it decreases some of the negative aspects of the metabolic responses to injury. These are thought to be associated with an arginine-induced growth hormone release.

Wound healing accelerated by Staphylococcus aureus.

While comparing the effects on wound healing of a heated scalpel with those of the cold scalpel, we discovered that inoculation of rat skin incisions with a strain of Staphylococcus aureus dramatically accelerated the gain in wound strength. The accelerating effect was evident four days postoperatively, was maximal at seven to ten days, and was still present at 28 days. The accelerating effect was correlated with the number of S aureus organisms introduced into the wound, and was found in conventional rats and rats germ free up to the time of monocontamination with S aureus. There was no evidence of infection on gross examination; on histologic examination an occasional microabscess was seen in some rats. There may be both local and systemic mechanisms underlying the S aureus accelerating effect. Seven strains of S aureus with varying characteristics demonstrated the wound-healing accelerating effect. In sharp contrast, Staphylococcus epidermidis (three strains), Staphylococcus hominis (one strain), and Pseudomonas aeruginosa (two strains) did not show this effect. The increases in wound healing due to S aureus were substantially greater than reported previously for any nutritional supplement, drug, or other chemical or physical agent.