Albert B. Deisseroth

Wild-Type Human p53 and a Temperature-Sensitive Mutant Induce Fas/APO-1 Expression

Fas/APO-1 is a cell surface protein known to trigger apoptosis upon specific antibody engagement. Because wild-type p53 can activate transcription as well as induce apoptosis, we queried whether p53 might upregulate Fas/APO-1. To explore this possibility, we examined human p53-null (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non-small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replication-deficient adenovirus expression construct containing the human wild-type p53 gene but not with vector alone, a marked upregulation (approximately a three-to fourfold increase) of cell surface Fas/APO-1 was observed by flow cytometry. Similarly, K562, cells stably transfected with a plasmid vector containing the temperature-sensitive human p53 mutant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 degrees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-143 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) analyses. Stably transfected K562 cells expressing temperature-insensitive, transcriptionally inactive p53 mutants (His-175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 occurred in the presence of cycloheximide, indicating that de novo protein synthesis was not required and suggested a direct involvement of p53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53.

Lethal pulmonary reactions associated with the combined use of amphotericin B and leukocyte transfusions.

Amphotericin B is used increasingly in high-risk patients with profound neutropenia and suspected sepsis. We have observed serious pulmonary reactions characterized by acute dyspnea, hypoxemia, and interstitial infiltrates on chest films in patients receiving amphotericin B and leukocyte transfusions. We reviewed 6 1/2 years of experience at the National Institutes of Health to determine whether this combination was associated with pulmonary toxicity not characteristic of either therapy alone. Amphotericin was used during 22 of 57 leukocyte-transfusion courses. Acute respiratory deterioration occurred during 14 (64 per cent) of these courses but in only two (6 per cent) of 35 courses without amphotericin (P less than 0.01). In seven cases, respiratory deterioration began during or immediately after amphotericin infusion, and it contributed to death in five patients. Diffuse intraalveolar hemorrhage was found when lung biopsy or autopsy was performed. Acute respiratory deterioration was not observed in comparably neutropenic patients given amphotericin but not leukocyte transfusions during the same period. It was mot common when amphotericin was begun with or after institution of daily leukocyte transfusions. Leukocyte transfusions may cause changes in the lungs that amplify the acute toxicity of amphotericin, thereby permitting severe pulmonary reactions.

Duration of empiric antibiotic therapy in granulocytopenic patients with cancer

Abstract Early initiation of empiric antibiotic therapy in febrile cancer patients has become established practice, but the appropriate duration of antibiotic therapy when no infectious source can be identified is unknown. The complications of broad-spectrum antibiotics argue for brief treatment, but the risk of an inadequately treated infection in the granulocytopenic patient favors longer therapy. We prospectively studied 306 episodes of fever and granulocytopenia in 143 patients with leukemia or solid tumor (age one to 33 years) with respect to the duration of empiric antibiotic treatment. Eligible patients (fever > 38 °C three times/24 hours or > 38.5 °C once, plus polymorphonuclear leukocytes 3 ) had an extensive diagnostic evaluation, including at least two preantibiotic blood cultures, and therapy was then started with a broad-spectrum antibiotic regimen— Keflin ® , gentamicin and carbenicillin (KGC). Initial evaluation failed to identify an infectious etiology for the fever in 142 of 306 (46 per cent) episodes. Fifty-six of 142 (39 per cent) of these fevers of unknown origin were associated with persistent granulocytopenia for more than seven days; in 33 of these, defervescence occurred while the patients received KGC. After seven days of empiric KGC therapy, the 33 patients with fevers of unknown origin who had become afebrile with empiric antibiotics but whose polymorphonuclear leukocytes remained less than 500/mm 3 were randomized to either continue or discontinue (dc) to receive KGC. The patients who continued to receive KGC until their polymorphonuclear leukocytes were more than 500/mm 3 had no infectious sequelae. However, in seven of 17 (41 per cent) of the patients randomized to dc KGC infectious sequelae developed (p = 0.007) within a median of two days of discontinuing KGC (two with fever which again responded to KGC therapy, and five with a documented infection [two ultimately fatal]). In none of the patients did a resistant microbial flora or superinfection develop. These data suggest that the patient with a fever of unknown origin who becomes afebrile during empiric antibiotic therapy may profit from continued therapy while granulocytopenia persists.

Localization of the human α-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

Abstract We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.

Prolonged complete remission following high dose chemotherapy of burkitt's lymphoma in relapse

Fourteen patients with American Burkitts lymphoma resistant to conventional chemotherapy were treated with high‐dose combination chemotherapy and intensive supportive care. Four patients died shortly after chemotherapy, 3 of an acute carditis. All ten remaining patients demonstrated tumor regression and 3 remain in prolonged complete unmaintained remission 29+, 19+, and 9+ months after treatment. These findings demonstrate that high‐dose chemotherapy will benefit some patients with Burkitts lymphoma unresponsive to conventional chemotherapy, but the medullary and extramedullary toxicity of this treatment strategy remains a formidable obstacle.

Interaction of nuclear proteins with an AP-1/CRE-like promoter sequence in the human TNF-α gene

The tumor necrosis factor α (TNF‐α) promoter contains an AP‐l/CRE‐like binding site, TGAGCTCA. AP‐1 elements generally transduce signals involving protein kinase C; the CRE site mediates a cAMP response, involving protein kinase A. Thus, this element has the potential to receive signals through divergent signaling pathways. Nuclear protein binding studies using extracts from THP‐1 monocytic cells treated with lipopolysaccharide (LPS), which stimulates, or dexamethasone (Dex) or pentoxifylline (PTX), which inhibit TNF production, respectively, suggest that two lowmobility complexes could be involved in regulation through this promoter region. PTX and Dex increase binding of both these complexes compared with untreated cells; approximately 2 hours after LPS induction, the upper complex becomes undetectable. This upper complex is composed of ATF2 (activating transcription factor 2, a cyclic AMP responsive element binding protein) homodimers; the lower is a heterodimer of jun/ATF2. LPS induces c‐jun and thus may enhance formation of jun/ATF2 complexes, which could be activating complexes. In this case, the simultaneous presence of both complexes, which would occur in the presence of Dex or PTX, could reduce the amount of TNF transcription through competitive binding. Through in vitro competitive binding studies comparing the binding affinities of the TNF promoter sequence and a consensus CRE, we further suggest how variation of endogenous binding sequences from consensus may be an important property for regulatory control of particular genes J. Leukoc. Biol. 56: 27–35; 1994.

Treatment of poor prognosis nonseminomatous testicular cancer with a “high‐dose” platinum combination chemotherapy regimen

A new intensive four drug combination chemotherapy regimen, termed PVeBV, consisting of cis‐platinum, vinblastine, bleomycin, and VP‐16, was administered to six previously untreated patients with poor prognosis advanced nonseminomatous testicular cancer and to four patients who had relapsed on primary platinum based regimens. The cis‐platinum was administered in 250 ml of 3% saline at twice the dose (40 mg/m2 IV days 1–5 every three weeks) used in other treatment schedules. All six previously untreated patients achieved a complete remission. Four achieved a complete remission with three cycles of PVeBV while the other two patients achieved a complete remission with an additional cycle of cisplatinum and VP‐16 at 200 mg/m2 IV × five followed by autologous bone marrow infusion. All four relapsed patients responded to PVeBV (two complete remissions and two partial remissions). There were no deaths associated with PVeBV therapy; however, myelosuppression was severe. There has been no renal toxicity (other than hypomagnesemia) observed with 35 cycles of high‐dose platinum therapy in previously untreated patients. These results indicate that PVeBV is a promising chemotherapy regimen for the treatment of poor prognosis testicular cancer patients. Furthermore, it appears that cis‐platinum can be administered at higher doses than previously used without an increase in renal toxicity if administered in hypertonic saline. The high‐dose cis‐platinum schedule, as used in PVeBV, warrants evaluation in other tumors which respond to standard‐dose platinum therapy.

Allogeneic bone marrow transplantation for refractory and recurrent low-grade lymphoma: the case for aggressive management.

PURPOSE To evaluate the role of allogeneic bone marrow transplantation (BMT) in recurrent low-grade lymphoma. PATIENTS AND METHODS Between 1989 and 1994, 10 patients with chemotherapy-refractory and recurrent low-grade lymphoma were treated with myeloablative therapy and allogeneic BMT. All patients had poor prognostic features and had been extensively pretreated. RESULTS Eight patients achieved a complete remission and none have relapsed at a median follow-up time of 816 days (range, 346 to 1,865). Two patients died of early complications. The actuarial survival and failure-free survival rates are both 80% +/- 12.6%. For surviving patients, the duration of the current remission exceeds that of any previous remission achieved. CONCLUSION These results compare favorably with those for autologous BMT. Allogeneic BMT offers considerable promise for the treatment of patients with poor-prognosis low-grade lymphoma. Its role should be further defined in prospective studies.

Allogeneic bone marrow transplantation for poor-prognosis lymphoma: Response, toxicity, and survival depend on disease histology

PURPOSE To evaluate outcomes and identify prognostic factors in allogeneic bone marrow transplantation in patients with end-stage lymphoma. PATIENTS AND METHODS Data were retrospectively analyzed of 64 patients (42 men and 22 women) 18 to 48 years of age with recurrent or refractory lymphoma who underwent allogeneic bone marrow transplantation from matched sibling donors (or in 1 case from a one antigen-mismatched relative) between May 1981 and July 1994. RESULTS Twelve patients survived free of disease. They were 8 of 15 with low-grade lymphoma (disease-free survival at 2 years 59% +/- 13%); 3 of 25 with lymphoblastic lymphoma (disease-free survival 17% +/- 8%); and 1 of 10 with diffuse small non-cleaved cell lymphoma (disease-free (10% +/- 9%). Survival and disease-free survival of patients with low-grade lymphoma were significantly superior compared to any other subgroup of patients (P <0.01). Only 2 patients with low-grade lymphoma had disease progression (9% +/- 9% actuarial risk at 2 years) as opposed to 5 of 15 with intermediate-grade lymphoma (39% +/- 14%), 9 of 25 with lymphoblastic lymphoma (28% +/- 9%), and 8 of 10 (80% +/- 13%) with diffuse small non-cleaved lymphoma. The actuarial risk for disease progression was significantly lower for patients with low-grade lymphoma than for any other histologic subgroup (P <0.02). It was significantly higher for those with diffuse small non-cleaved cell lymphoma than for other histologic subgroups (P < or = 0.003). CONCLUSIONS Allogeneic bone marrow transplantation is an effective procedure in patients with refractory low-grade lymphoma. It results in long-term remissions and should be considered in younger patients with recurrent disease who have a matched sibling donor. The late recurrence in 1 patient indicates the necessity of continued follow-up. A small fraction of patients with end-stage intermediate- and high-grade lymphoma can obtain prolonged disease-free survival, but recurrence and regimen-related toxicity remain major problems. The results could be improved by the development of conditioning regimens with less toxicity and by the use of bone marrow transplantation earlier in the course of the disease.

A temperature-sensitive mutant of human p53.

We discovered that the human ‘hot‐spot’ p53 mutant 143Ala is temperature sensitive for the binding of two DNA elements and for mediating the transcription of a downstream luciferase reporter gene. At 32.5 degrees C, 143Ala possesses strong DNA binding ability. In addition, its transcriptional activities are stronger than those of wild‐type p53. At 37.5 degrees C, however, both the mutants DNA binding and transcriptional activation functions are greatly reduced or abolished. The activity differences correlate with the presence of two conformational states of p53, which are recognized by monoclonal antibodies: at 32.5 degrees C, 143Ala is recognized by PAb1620, which is specific for the wild‐type conformation, whereas at 37.5 degrees C, 143Ala is almost undetectable by PAb1620‐mediated immunoprecipitation. Although 143Alas binding to p53 DNA recognition elements and its activation of reporter gene transcription at 32.5 degrees C is markedly higher than that of the wild‐type p53, 143Ala inhibited proliferation less robustly than wild‐type p53 and it did not increase inhibition of ras‐induced focus formation. These results indicate a partial correlation between the events involved in transcriptional activation and events involved in the proliferation suppression of p53. 143Ala therefore will be of use in dissecting the relationship between the structure of p53 and its different cellular functions.