Intestinal cell apoptosis and Bcl-2 expression.

Programmed cell death (apoptosis) is a normal characteristic of cells with a limited life span like the enterocyte and the usual mode of death for proliferative crypt cells subjected to radiation or chemotherapy. The Bcl-2 proto-oncogene is considered a major regulator of apoptosis. We investigated the relationship of enterocyte apoptosis and Bcl-2 expression in rat intestine and tissue culture cells. Fragmentation of DNA and levels of Bcl-2 transcripts were evaluated in rat enterocyte fractions of the crypt-to-villus axis of differentiation and in IEC tissue culture cells. A low percentage of isolated nuclei from each enterocyte fraction showed features of DNA fragmentation, including crypt cells. Detectable DNA fragmentation was seen in IEC cells only when cells were subjected to long-term confluent culture conditions. Bcl-2 mRNA was not detected in isolated rat intestinal cells but was detected in IEC cells where its level increased with serum deprivation and long-term culture. We conclude that increased Bcl-2 expression may be important in rescue of proliferative enterocytes subjected to stress.

Laminin receptor expression in rat intestine and liver during development and differentiation.

BACKGROUND/AIMS: Studies have identified a 67-kilodalton high-affinity laminin receptor (LR) whose expression has also been related to development, differentiation, and neoplastic transformation. The relationship of the 67-kilodalton LR to hepatic and enterocyte development and to enterocyte differentiation was investigated. METHODS: LR messenger RNA (mRNA) was identified using a complementary DNA isolated from a rat crypt cell library. LR and integrin (alpha 6, beta 1, and beta 4) expression by rat intestinal crypt cells was compared with that of the more differentiated villus cells using Northern blotting. Developmental differences in LR expression were studied in fetal and neonatal rats. The pattern of LR expression in fetal and adult rat intestines was examined further by in situ hybridization. RESULTS: LR mRNA levels were highest in fetal liver and intestine and adult rat crypt cells. LR mRNA levels were 9-10 times greater in crypt than in villus cells. Integrin subunit expression differed little between crypt and villus cells. Nascent transcription studies showed that the proportion of newly transcribed LR mRNA per total RNA synthesized was similar for crypt and villus cells, suggesting posttranscriptional control of LR mRNA levels in villus cells. CONCLUSIONS: Increased LR mRNA expression is a feature of the fetal intestine and of the undifferentiated, mitotically active crypt cells.

Changes in ribosomal protein and ribosomal RNA synthesis during rat intestinal differentiation.

Subtraction hybridization studies, used to identify genes involved in the control of enterocyte proliferation and/or differentiation, allowed detection of a clone shown to have homologies with rat, chicken, and human acidic ribosomal phosphoprotein P1. Since increases in P1 transcript have been associated with intestinal malignancy, we explored the relationship of P1 and other ribosomal proteins to normal intestinal proliferation and differentiation. Male rats were used to prepare enterocytes as isolated cell fractions representative of the crypt to villus axis of differentiation. Total RNA was extracted from pooled cell fractions and evaluated for mRNA and rRNA steady-state levels. Nuclei were prepared from isolated enterocytes, and nuclear runoff studies were performed to estimate rates of nascent transcription. The P1 complementary DNA from the crypt cell library detected a mRNA of 650 base pairs which showed approximately 8-fold greater steady-state levels in crypt than in villus cells. Similar crypt specificity was also noted for mRNAs coding for elongation factor EF-12 and for ribosomal proteins P0, P1, and S6 (using clones from Y-L. Chan and I. G. Wool). In contrast, 28S rRNA steady-state levels did not differ between villus and crypt, indicating that ribosomal content had remained constant. In situ hybridization studies confirmed the predominant crypt localization of P1 mRNA. Nascent transcription rate studies showed that the proportion of newly synthesized P1 mRNA to total RNA was the same for the villus and crypt, suggesting that the lower content of villus P1 mRNA may be due to increased degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in cell cycle characteristics among patients with acute nonlymphocytic leukemia.

The proliferative characteristics of myeloid leukemias were defined in vivo after intravenous infusions of bromodeoxyuridine (BrdU) in 40 patients. The percentage of S-phase cells obtained from the biopsies (mean, 20%) were significantly higher (P = .00003) than those determined from the bone marrow (BM) aspirates (mean, 9%). The post-BrdU infusion BM aspirates from 40 patients were incubated with tritiated thymidine in vitro. These double-labeled slides were utilized to determine the duration of S-phase (Ts) in myeloblasts and their total cell cycle time (Tc). The Ts varied from four to 49 hours (mean, 19 hours; median, 17 hours). Similarly, there were wide variations in Tc of individual patients ranging from 16 to 292 hours (mean, 93 hours; median, 76 hours). There was no relationship between Tc and the percentage of S-phase cells, but there was a good correlation between Tc and Ts (r = .8). Patients with relapsed acute nonlymphocytic leukemia (ANLL) appeared to have a longer Ts and Tc than those studied at initial diagnosis. A subgroup of patients at either extreme of Tc were identified who demonstrated clinically documented resistance in response to multiple courses of chemotherapy. We conclude that Ts and Tc provide additional biologic information that may be valuable in understanding the variations observed in the natural history of ANLL.

Analysis of cell cycle characteristics and course of the disease in ANLL.

The cell cycle characteristics of a newly diagnosed patient with acute nonlymphocytic leukemia (ANLL) were studied by using intravenous bromodeoxyuridine (BrdU) and our previously described "double-label" technique. The percentage of S-phase cells in the bone marrow (BM) biopsy were 25% compared to 7% from the simultaneously obtained BM aspirate. The duration of S-phase (Ts) was determined to be 4 hr and the total cell cycle time (Tc) was 16 hr. We demonstrated that the actual clinical course of this patient's illness corresponded well with the course predicted on the basis of these cell cycle measurements. Although he achieved aplasia in response to two successive courses of induction chemotherapy, leukemic cells repopulated the marrow, producing a rapidly rising PB white blood cell (WBC) count with a T1/2 of approximately 20 hr each time. It is likely that the resistance of this patient's leukemia to therapy was a result of the rapid proliferative rate of his leukemic cells and not due to the inability of chemotherapeutic agents to kill a large number of cells. Since the measurements can be completed within 48 hr, measuring Ts and Tc will provide a better understanding of the biological differences that exist between patients with ANLL.

A new method for studying cell cycle characteristics in ANLL using double-labeling with BrdU and 3HTdr.

Ten patients with acute nonlymphocytic leukemia (ANLL) received bromodeoxyuridine (BrdU) at 100 mg/M2 intravenously over 1 h. BrdU is incorporated into the DNA by S-phase cells and was detected by using a monoclonal anti-BrdU antibody in the bone marrow aspirate (BM) and biopsy specimens obtained at the end of the infusion. Additionally, BM was incubated in vitro with tritiated thymidine (3HTdr) and processed by our previously described double-label method. This allowed us to measure the duration of S-phase (Ts) and total cell cycle time (Tc) of myeloblasts. Data revealed a higher number of S-phase cells from biopsies (21%) than BM (5%). The Ts ranged from 9 to 35 h and Tc ranged between 36 and 152 h in different patients. Using this method, data are available within 48 h and if shown to be clinically relevant, may be useful for prospective planning of therapy in individual patients.

Proliferative characteristics of acute nonlymphocytic leukemia in vivo.

The proliferative characteristics of myeloid leukemias were defined in vivo following intravenous bromodeoxyuridine (BrdU). Fifteen patients received a 2-hour infusion of BrdU. A monoclonal anti-BrdU antibody was used to detect the in vivo incorporation of BrdU by S-phase cells. The percentage of S-phase cells obtained from the biopsies (mean 17.3%) was significantly higher (p = 0.00001) than the percentage determined from the aspirates (7.8%). It is concluded that the true estimate of S-phase cells can only be obtained from biopsies following in vivo labeling of cells synthesizing DNA. The persistence of BrdU-labeled cells in follow-up studies can be used to recognize 'residual leukemia', and the subsequent fate of these cells can be defined in vivo.

Effect of portal vein occlusion on liver blood flow in normal and cirrhotic dogs.

The purpose of this study was to demonstrate that galactose clearance (GC) can measure acute changes in liver blood flow (LBF) in normal and cirrhotic dogs. Ten dogs were studied. GC was measured preop. At laparotomy, GC, hepatic artery (HA) flow, portal vein (PV) flow, and cardiac output (CO) were measured at baseline, 50% portal vein occlusion (PVO), and portal vein release. HA and PV flows were measured using a flow probe (FP). Common bile duct ligation was then performed to cause cirrhosis and all measurements were repeated in 7 weeks. Statistical analyses showed that on PVO in both normal dogs (n = 10) and cirrhotic dogs (n = 5) the GC, HA flow, and CO were significantly different from their baseline values. In both groups PVO caused HA flow to increase, thus keeping FP-LBF unchanged while GC-LBF was significantly reduced compared to baseline. The possible explanations for this are discussed in the text. PVO also caused a significant reduction in CO due to splanchnic pooling in both normal and cirrhotic dogs. In both groups PVO results in an increased percentage of CO going to FP-LBF, while the percentage of CO going to GC-LBF remains unchanged. We conclude that GC can measure acute changes in LBF caused by a 50% PVO in both normal and cirrhotic dogs.

Blunt liver trauma at the Sunnybrook Regional Trauma Unit.

Between June 1, 1976 and Mar. 31, 1983, the Sunnybrook Medical Centre Regional Trauma Unit in Toronto, Ont., admitted 145 patients with liver trauma; of these, 141 (97%) had sustained blunt liver trauma. Of 113 patients who underwent open peritoneal lavage, 112 had a true-positive lavage. Resuscitation was successful in 137 patients and 134 of these underwent laparotomy. Seventy-nine (59%) of the 134 patients required only minor surgical treatment; the other 55 (41%) required major surgical procedures. The overall mortality was 32% (47 of 145). Eight patients died during resuscitation but only one of them died of liver hemorrhage. Of the 39 patients who died after admission, the cause of death was head injury in 22, while 6 died of liver hemorrhage and 11 of other causes. Overall, liver hemorrhage was the cause of death in 15% of cases (7 of 47).

Impact of therapy for differentiated carcinoma of the thyroid: an analysis of 706 cases.

A retrospective analysis of clinical and pathological data was conducted on 706 patients (514 females and 192 males) treated for differentiated thyroid carcinoma at The University of Texas M. D. Anderson Hospital and Tumor Institute at Houston from 1951 to 1975 and followed to 1981. The histological diagnoses were mixed papillary/follicular carcinoma (66.7%), papillary carcinoma (14.6%), follicular carcinoma (15.3%), or Hurthle cell carcinoma (3.4%). Patients diagnosed before the age of 40 yr lived significantly longer than those diagnosed over the age of 40 yr, and females lived longer than males. According to survival analyses and disease-free intervals, the order of increasing aggressiveness of the tumors was papillary, mixed, follicular, and Hurthle cell. Total thyroidectomy was associated with longer disease-free intervals and fewer recurrences. The 136 patients who received ablative 131I after surgery had fewer recurrences than a matched group who did not, but the disease-free interval and survival rate showed no significant difference. Further classification showed that patients with follicular and mixed tumors, and those who underwent total thyroidectomy benefited from 131I. There were 78 deaths attributed to thyroid cancer in the whole group. Approximately two thirds occurred in the first 10 yr after diagnosis. In conclusion, total thyroidectomy is recommended, when feasible, for differentiated thyroid cancer, followed by ablative 131I therapy, at least for follicular and mixed varieties.

131I therapy in differentiated thyroid carcinoma: M. D. Anderson Hospital experience.

The therapeutic response and survival rates of 352 patients with differentiated thyroid carcinoma who had received radioactive iodine therapy since 1951 were studied. Of these patients, 72% had metastases to the cervical lymph nodes, lungs, bone, or other viscera. Of all patients, 70% showed complete remission, and 30% showed partial response or recurrence of disease or both. Patients with metastases before therapy had a higher incidence of recurrence than those who had localized thyroid disease (32.4% vs. 15.1%). Of these, 97 patients had recurrent disease. In 24 of these patients, the recurrent tumor failed to take iodine, indicating change in its iodine-concentrating characteristics. Of the 352 patients, 44 (12.5%) died of progressive thyroid carcinoma, and all were over 40 years of age at the time of initial diagnosis. Mean survival of patients with metastatic disease who were over 40 years of age at initial diagnosis was significantly lower than that of patients under 40 years of age (6.2 years vs. 11.5 years). Patients with recurrent metastases unresponsive to surgery or radioactive iodine therapy were treated with palliative radiotherapy or chemotherapy or both. They respond poorly and died within a few months.