Diminished molecular response to doxorubicin and loss of cardioprotective effect of dexrazoxane in Egr-1 deficient female mice.

Doxorubicin (DOX) and VP16 are DNA topoisomerase II inhibitors yet only DOX induces an irreversible cardiotoxicity, likely through DOX-induced oxidative stress. Egr-1 is overexpressed after many stimuli that increase oxidative stress in vitro and after DOX-injection into adult mice in vivo. To investigate Egr-1 function in the heart, we compared the molecular and histological responses of wild type (+/+) and Egr-1 deficient (-/-) female mice to saline, DOX, VP16, the cardioprotectant dexrazoxane (DZR), or DOX+DZR injection. DOX, and to a lesser extent VP16, induced characteristic increases in cardiac muscle and non-muscle genes typical of cardiac damage in +/+ mice, whereas only beta-MHC and Sp1 were increased in -/- mice. DZR-alone treated +/+ mice showed increased cardiomyocyte transnuclear width without a change to the heart to body weight (HW/BW) ratio. However, DZR-alone treated -/- mice had an increased HW/BW, increased cardiomyocyte transnuclear width, and gene expression changes similar to DOX-injected +/+ mice. DZR pre-injection alleviated DOX-induced gene changes in +/+ mice; in DZR+DOX injected -/- mice the increases in cardiac and non-muscle gene expression were equal to, or exceeded that, detected after DOX-alone or DZR-alone injections. We conclude that Egr-1 is required for DOX-induced molecular changes and for DZR-mediated cardioprotection.

Morphometry and muscle gene expression in hypertrophied hearts from polyomavirus large T antigen transgenic mice.

Transgenic mice expressing polyomavirus large T antigen (PVLT) in cardiomyocytes develop a cardiac hypertrophy in adulthood. Morphometric analysis identified cardiomyocytes enlarged up to ninefold in cross-sectional area in the adult transgenic hearts compared with normal age-matched nontransgenic hearts. Most enlarged cardiomyocytes were found in the subendocardium, whereas normal-sized cardiomyocytes were localized to the midmyocardium. Transgenic hearts did not express detectable skeletal muscle actin mRNA or protein, or skeletal troponin I isoform mRNA. Some, but not all, transgenic hearts expressed an increase in the beta-myosin heavy chain mRNA. All five transgenic mice tested had increased expression of atrial natriuretic factor (ANF) mRNA. Whereas normal hearts expressed three myosin light chain proteins of 19, 16, and 15 kDa, we found that the 19-kDa myosin light chain was not observed in the transgenic hearts. We conclude that adult, PVLT-expressing, transgenic mice developed enlarged cardiomyocytes with an increase in beta-myosin heavy chain and ANF mRNA expression, but a widespread skeletal isoform usage was not present in these transgenic mice. The adult transgenic hearts thus display histological and molecular changes similar to those found in hypertrophy induced by a pressure overload in vivo.

Characterization of the tissue regression process in the uterus of older mice as apoptotic by the presence of Tp30, an isoform of terminin.

Whether old uteri that have undergone involution do so by an apoptotic mechanism was examined by the presence of known biochemical and morphological markers for programmed cell death. Terminin, a protein identified by an unique monoclonal antibody, has three forms, Tp-90, Tp-60, and Tp-30: Tp-90 (the 90 kDa form) is only present in growing and quiescent non-growing cells; Tp-60 (the 60 kDa form) is found in senescent cells; and finally, Tp-30 (the 30 kDa form) is found in cells committed to apoptotic death. Biochemical analysis of a protein, Tp30, previously identified as a marker for the commitment to programmed cell death, was performed with both young (5-month-old) and old (24-month-old) C57BL/6J mouse uteri. In addition to biochemical analysis of Tp30 presence in uterine tissue, propidium iodide (PI) staining and DNA framentation by nick-end labelling with fluorescence-conjugated UTP were used to characterize apoptosis-related changes in the chromatin organization of the nucleus. Results indicate that within the old uterus Tp-30 is indeed detected in the tissue extracts and was the major terminin band, while Tp-90 and Tp-60 were the major bands observed in extracts of the younger mouse uterus. The presence of Tp30 in the older uterine tissue suggests that the tissue regression which has occurred in the uterus of older mice may be apoptotic in nature. This suggestion is further supported by the demonstration of increases in the number of cells showing apoptotic morphology, i.e. positive staining with UTP reflecting the presence of nuclei with nicked DNA, localized exclusively in the uterine stroma of older females. The presence of DNA fragmentation, as reflected by UTP staining, was virtually absent from the young uteri. These data suggest that apoptosis may be a part of the cellular mechanism contributing to the regression of uterine tissue in the older female during involution, appearing as an age-dependent event.

Proliferative activity at colonic anastomoses as determined by statin. A nonproliferation-specific nuclear protein.

PURPOSE: One theory of anastomotic recurrence in large bowel carcinoma is that epithelial hyperplasia at the suture line causes metachronous carcinoma. METHODS: S44, a monoclonal antibody directed against statin, a nuclear protein expressed in quiescent cells, was used to determine whether the anastomosis represents an area with a high proliferation rate. During follow-up colonoscopic examination of patients who had undergone previous resection for colorectal carcinoma, biopsies were taken from the anastomotic site and from the mucosa 10 to 15 cm from the anastomosis. One side of 10 well-oriented crypts was counted for each patient with the number of nuclei positive for statin being determined by the presence of dark brown reaction product. RESULTS: The average percentages of statin-positive cells varied between 19.4 and 44.4 (average, 31.3 +/- 6.5) for the normal mucosa and 22.8 to 35.1 (average, 29.98 +/- 3.67) for the anastomotic mucosa. The differences were not significant. There were no differences between those patients in whom the postoperative time elapsed was two years or less and those greater than two years. CONCLUSION: This study is unique in that the proliferative activity at the site of colonic anastomosis was determined in a clinical setting, and patients in which the anastomoses were created anywhere from 1 to 14 years earlier were included. Using S44 as a marker, this study does not support the theory that suture line recurrence is a result of an enhanced proliferation rate.

Intramural leiomyosarcoma of the dorsal pedal vein: a clinical mimicry of ganglion.

A 75-year-old woman presented with a ganglion-like nodule on the dorsal aspect of the right foot. A 2.5 x 1.5 cm, saccular and malleable tumor, that was in continuity with the dorsal venous arch, was completely resected. It was characterized by a diffuse intramural and circumferential, low grade, malignant, smooth muscle proliferation with an aneurysmal-like luminal space. No endoluminal or periadventitial invasive neoplastic component was present. The patient had no evidence of disease at 58-month follow-up. This is the first reported case of venous leiomyosarcoma in the foot. Furthermore, the intramural confinement of neoplastic growth is a unique observation.

Immunohistochemical expression of mutant p53 oncogene in transitional mucosa adjacent to human colon cancer.

The purpose of this study was to analyze the expression of a mutant (MUT) p53 oncogene protein in the mucosal crypts adjacent to a human colon cancer. Five 1-cm mucosal segments were taken from the surgical specimens over a 5-cm distance from the tumor. Immunohistochemistry was performed using a monoclonal antibody (Ab3) to the MUT p53 and examination by light microscopy. The mean % labelling index (LI) of 10 crypts/cm segment was determined by image analysis. The LI for the entire crypt length for the first cm segment was 33.51 +/- 4.2 and for the second cm segment was 29.26 +/- 5.4 (p < 0.02). Due to the unequal distribution of the label within the crypt length, it was divided into halves so that the LI of these levels could be determined. The LI for the upper and lower crypt levels for the first cm segment were 28.67 +/- 3.2 and 78.23 +/- 4.6 (p < 0.01); for the second segment, the LI were 22.0 +/- 5.1 and 68.66 +/- 4.7 (p < 0.01). No expression of MUT p53 nuclear protein was noted distally at 3-5 cm. The localization of MUT p53 protein product to the crypt stem cell nucleus supports the contention that a malignant field change exists in the transitional mucosa adjacent to a human colon cancer.

Temporal relationships of statin and terminin expression in ventral lobe of rat prostate following castration.

The purpose of this study was to determine the temporal relationships, in the rat prostate following castration, the expressions of terminin, a cytoplasmic marker for senescence, and, statin, a nuclear marker for cell quiescence and senescence. The presence of these two proteins was determined at 0, 1, 2, 4, 8, 24 and 48 hours in the ventral lobe of the prostate following castration. Immunofluorescence techniques for double labelling were used and assessed with confocal microscopy. At 0 hour the mean % labelling index (LI) for terminin was 0% and 98% for statin. One hour following castration a complete reversal of expression of these two markers occurred indicating that the terminin marker is expressed at the start of programmed cell death or apoptosis. The mean % LI for terminin at 1, 2, 4, 8, 24 and 48 hours following castration were 54, 82, 63, 39, 44 and 41% respectively. The mean % labelling index of statin remained at zero during these time intervals. It is concluded that following castration, the prostatic ventral lobe exits from the quiescent phase to reenter cell cycle traverse. This is coupled by the loss of statin and the expression of the cytoplasmic marker terminin at the start of programmed cell death prior to the appearance of any histologic features of apoptosis.

The differential expression of statin in the nuclei of human colonic crypts adjacent to a cancer: an immunohistochemical study.

Statin, a non-proliferation-specific nuclear antigen, was used here to assess the colonic crypt kinetics of the mucosa bordering a human colon cancer. Mucosal strips adjacent to a colon cancer obtained from operative specimens were immediately cut into five one cm segments and stored in liquid nitrogen. An immunohistological technique using the statin antibody as a nuclear marker was used to determine the labelling indices of the non-cycling compartment at the varying distances. Optical density measurements of the nuclear reaction product served to objectively identify the statin-positive nucleus. The results indicate that there is a statistically significant reduction (P < 0.0001) in the statin-positive labelling index in the entire crypt length for a distance of three cms. The division of the entire crypt into four levels (A, B, C and D) demonstrates that this effect is principally due to the upward extension of the statin-negative cell mass into levels B and C with a corresponding decrease in the labelling index of the statin-positive nuclei in these levels. The in vivo expression of nuclear statin demonstrates its usefulness in accurately determining the size of the non-proliferative compartment in the human colonic crypt adjacent to a colon cancer.

The care of patients with colorectal polyps that contain invasive adenocarcinoma. Endoscopic polypectomy or colectomy?

BACKGROUND: The appropriateness of resection in patients from whom polyps with invasive adenocarcinoma were excised has been questioned. METHODS: To determine the results of this policy, the authors reviewed the outcome of 42 patients from whom 44 such polyps were removed. Each polyp was categorized for the level of invasion according to the classification of Haggitt. RESULTS: Level 1 invasion was found in 27%; level 2, in 9%; level 3, in 11%; level 4, in 39%; and uncertain, in 14%. The histologic grade was well differentiated in 48% of patients and moderately differentiated in 52%. No polyps contained poorly differentiated adenocarcinoma; lymphatic and vascular invasion were not encountered. Excision was judged complete in 23 patients; 11 underwent resection, and in none was residual adenocarcinoma identified. In 14 patients, margins could not be evaluated; of 12 patients who underwent resection, residual adenocarcinoma was found in 1. Of the seven patients with positive margins who underwent resection, residual adenocarcinoma was found in only two. In the resected specimens in which residual carcinoma was encountered, all original lesions were designated level 4. None of the patients treated by polypectomy alone has experienced a recurrence at a mean follow-up time of 66 months (range, 12-152 months). CONCLUSIONS: The authors conclude that only patients with level 4 invasion require resection.

Proliferative activity of colonic mucosa at different distances from primary adenocarcinoma as determined by the presence of statin: a nonproliferation-specific nuclear protein.

The field change is one hypothesis concerning the development of colorectal carcinoma. Removal of a carcinoma without its entire surrounding altered mucosa may result in the development of a recurrence. S44, a monoclonal antibody directed against statin, a nuclear protein expressed in nonproliferating cells in either a quiescent or senescent state, was used to determine the rate of cell growth in colorectal mucosa at different distances from carcinomas. The specimens of 18 patients undergoing resection of a colorectal carcinoma were immediately opened after operation, and strips of mucosa were taken at distances of 1 cm, 5 cm, and 10 cm from the carcinoma. For each location, 10 longitudinally oriented crypts were evaluated for statin-positive cells identified by the presence of a dark brown peroxidase-conjugated antibody reaction product. The average percentage of statin-positive cells per crypt was significantly lower at a 1-cm distance from the carcinoma compared with the mucosa located 5 and 10 cm from the carcinoma (20.89 +/- 4.33 at 1 cm, 32.41 +/- 5.27 at 5 cm, and 34.23 +/- 6.45 at 10 cm). None of the calculated parameters showed any significant difference between the 5-cm and 10-cm locations. The fact that the proliferation rate of the mucosal cells returns to the normal level at 5 cm from the margin of the carcinoma suggests that cells located within this distance still retain proliferative potential even though they are morphologically indistinguishable from their normal counterparts. We conclude that failure to remove this transitional, potentially proliferative mucosa may result in subsequent development of anastomotic or perianastomotic recurrences.

The value of nuclear morphometry in the management of patients with colorectal polyps that contain invasive adenocarcinoma.

Haggitt's classification is a useful guide in the management of patients with large bowel polyps which contain invasive adenocarcinoma in that patients with levels 1 to 3 require no operation. Nuclear morphometry has been shown to be a useful prognostic discriminant for patients with invasive carcinoma of the large bowel. The nuclear shape factor of 44 polyps with invasive carcinoma was studied to determine whether this parameter was of value to define those patients with Haggitt level 4 who should have a resection. The shape factor of 50 interphase nuclei was obtained through the use of image analysis by tracing the nuclear profiles as digitized on a video screen. The nuclear shape factor was defined as the degree of circularity of the nucleus, a perfect circle recorded as 1.0. Our previous experience showed a nuclear shape factor greater than 0.84 was associated with a poor outcome. The overall mean shape factor was 0.71 (0.59-0.85). There was a tendency for the patients with residual disease to have values in the upper range. Our findings suggest that nuclear morphometry fails to add any predictive information in this clinical situation.

Modulation of nuclear statin expression in rat thyroid follicle cell following administration of thyroid stimulating hormone.

This study was designed to examine the state of proliferation in the rat thyrocyte following the administration of thyroid stimulating hormone (TSH). An immunohistochemical technique involving the use of a monoclonal antibody to statin, a nonproliferation-specific nuclear antigen, was developed to measure the subpopulation of cells that have ceased to divide. Following the random assignment of young male Sprague-Dawley rats into various groups, the rats in the control group received a single intraperitoneal (i-p) injection of normal saline, whereas the experimental groups received single i-p injections of TSH at doses of 0.25, 0.50, and 1.0 IU, respectively. All rats were subsequently sacrificed in groups of three at 1, 2, 4, and 24 hours. The statin antibody label was readily identified within the follicle cell nucleus. Results revealed a statistically significant transient decrease in the mean percent statin-positive nuclei in the TSH-treated groups. The time- and dose-dependent effect of TSH was maximal at 2 hours and no longer discernible at 24 hours. A second experiment involving the chronic administration of TSH (i-p 0.25 IU twice daily) resulted in a cumulative response with a statistically significant progressive decrease in the mean percent of statin-positive nuclei at 5 and 10 days, returning to near normal values 5 days following the cessation of treatment. Determination of the nuclear optical density of the statin reaction product by image analysis techniques revealed that a single injection of TSH resulted in a rapid disappearance of the statin nuclear protein. This result suggests that the disappearance of statin in the nucleus appears to reflect the event of cells leaving the nondividing quiescent state to resume the cell cycle traverse following the administration of TSH. The disappearance of statin appears as an early nuclear event that parallels the earliest known cytoplasmic pinocytotic response to TSH in the rat thyroid follicle cell.

Histochemical localization of Statin--a non-proliferation-specific nuclear protein--in nuclei of normal and abnormal human thyroid tissue.

Surgical specimens of 29 human thyroid masses, both benign and malignant, were examined by means of a novel monoclonal antibody immunoreactive to statin, which is expressed only in quiescent G0 cells. The nuclei of normal thyroid follicle tissue together with nodular goitres and follicular adenomata had similar labelling indices of 96 +/- 2.67, 95 +/- 2.43 and 94 +/- 1.98 respectively. By contrast the labelling indices of papillary and undifferentiated thyroid malignancies were 82 +/- 3.05 and 15.2, respectively. These results indicate that normal thyroid tissues as well as benign thyroid tumors have similar non-proliferative activities. The differentiated papillary cancers have a smaller non-cycling compartment, the smallest being present in the most biologically aggressive undifferentiated thyroid cancer. The immunohistological evaluation of statin in the nuclei of human thyroid malignancies correlates with their biological behaviour in an inverse relationship.

Rumenectomy-induced proliferation in duodenal villous epithelium is mechanistically related to the disappearance of statin, a non-proliferation-specific nuclear protein.

We undertook this study to determine the effect of rumenectomy (a known cause of duodenal crypt cell hyperplasia) on the epithelial growth kinetics of the crypt-villus axis in rat duodenum. Ten rats were randomly assigned to control (gastrotomy) and experimental (rumenectomy) groups. After 14 days rats were sacrificed and representative sections were stained with the monoclonal antibody to statin, a non-proliferation-specific protein, by the immunoperoxidase procedure. In the control group, the mean percentages of statin-positive cells in the proximal duodenum, distal duodenum, proximal jejunum, and distal jejunum were 79 +/- 8.5, 79.5 +/- 5.7, 85 +/- 1.4, and 83.5 +/- 0.7, respectively. In the rumenectomy group, statin-positive nuclei were found in the region of the villous apices only, and the corresponding values for the above four areas were 26.2 +/- 4.9, 24.5 +/- 3.5, 31.7 +/- 4.5, and 80.5 +/- 2.1. Except for distal jejunum, the differences in statin expression in the control and experimental groups were significant (p less than 0.001). Rumenectomy leads to the disappearance of statin from the villous column cells of the duodenum and proximal small bowel. The lack of expression of statin in the rumenectomy group documents the potential usefulness of this measure in future studies in neoplasia were understanding of the proliferative status is of crucial importance.

Pagetoid carcinomatous involvement of the penile urethra in association with high-grade transitional cell carcinoma of the urinary bladder.

We present a case of pagetoid carcinomatous involvement of the penile urethra and periurethral glands following cystoprostatectomy for high-grade transitional cell carcinoma of the urinary bladder. Previously reported cases with involvement of the urethra are reviewed. This condition is believed to be a rare variant of transitional cell carcinoma in situ.

Nuclear shape as a prognostic discriminant in colorectal carcinoma.

In search for a more reliable prognostic discriminant, a retrospective analysis of 100 cases of colorectal carcinoma having undergone curative resection and followed for at least 5 years were assessed by nuclear morphometry. Each case was staged according to the Dukes' classification as well as graded histologically. For all patients in this series, the perimeter, area, and nuclear shape factor of 50 interphase nuclei were determined for each carcinoma. The information was obtained through the use of an image analysis system by tracing the nuclear profiles (magnification 1000x) as digitized on a video screen. The nuclear shape factor was defined as the degree of circularity of the nucleus, a perfect circle recorded as 1.0. A nuclear shape factor greater than 0.84 was associated with poor outcome. Multiple regression models showed that the single nuclear parameter of the shape factor was the most highly significant predictor of survival (P less than 0.0001). This variable remained highly significant even when corrected for sex, age, histologic grade, and Dukes' classification. These findings indicate that a nuclear shape factor greater than or equal to 0.84 as determined by nuclear morphometry is an independent morphometric nuclear variable of great importance in the prognosis of large bowel carcinoma.

Parathyroid cyst: diagnosis and treatment of an unusual surgical problem.

A case of parathyroid cyst is reported in which the diagnosis was suggested when watery, clear fluid was aspirated from a mass found in the anterior region of the neck of a 34-year-old woman on routine medical examination. The diagnosis was confirmed by measurement of the parathormone content in the cyst fluid and by histologic examination of the cyst wall. Although rare, parathyroid cyst should be considered in the differential diagnosis of cysts in the anterior compartment of the neck. Surgery has been the usual treatment of such cysts, but several reports have been published in which repeated aspiration resulted in the disappearance of the cyst. If conservative treatment of a parathyroid cyst is unsuccessful, the cyst should be removed surgically.

Enhanced carcinogenesis in the proximal small intestine of the rat following rumenectomy.

Various techniques have been used to enhance carcinogenesis in experimental animals. This study examines the effects of the excision of the forestomach (rumen), a squamous epithelial pouch of the rat, on the incidence and distribution of intestinal neoplasms induced by 1,2-dimethylhydrazine (DMH). Thirty-four male Sprague-Dawley rats were randomly assigned to control and experimental groups. The experimental group was subjected to an excision of the rumen while the control group underwent a gastro-rumenotomy and closure. Following a two week recovery period, each animal was weighed and injected with DMH (20 mgm/kg body wt) on a weekly basis for 22 weeks. At 24 weeks, the 32 surviving rats were sacrificed and the number, location, and histology of the neoplasms in the intestinal tract of each rat were noted. Rumenectomy resulted in a statistically increased incidence of neoplasms in the proximal small bowel (mean of 1.3 +/- 0.01 neoplasms/rat) when compared with the control group (mean of 0.1 +/- 0.2 neoplasms/rat) (p less than 0.001); but did not influence the incidence, distribution, or histology of colonic neoplasms between control and experimental animals. All neoplasms of the proximal small bowel when examined histologically were classified as invasive adenocarcinomas. The colon contained adenomata and carcinoma in situ, as well as adenocarcinomas. It is therefore concluded that excision of the rumen of the rat stomach selectively promotes malignant formation in the proximal small bowel following repeated injections of DMH.

Down-regulation of insulin binding by human and rat hepatocytes in primary culture: the possible role of insulin internalization and degradation.

Insulin binding and down-regulation were studied in primary cultures of human and rat hepatocytes. Equilibrium binding characteristics were similar in the two species, with a curvilinear Scatchard plot compatible with binding sites of high and low apparent affinities. The dose-response curve for insulin stimulation of glycogen synthesis coincided with the dose-occupancy curve of the low affinity sites; a maximal biological effect was reached at 50% occupancy. Exposure of rat hepatocytes to 2 X 10(-9) M insulin for 24 h produced a 48% decrease in binding capacity due to decreases in both types of binding sites and a 50% decrease in maximal insulin stimulation of glycogen synthesis. After exposure to the same insulin concentration human cells had an 83% decrease in maximum binding capacity, due exclusively to a complete loss of low affinity sites, and a total suppression of insulin stimulation of glycogen synthesis. In both species there was a biphasic relation between degradation and binding: over the range of insulin concentration producing binding mainly to high affinity sites degradation increased slowly as binding increased; with higher insulin concentrations and saturation of high affinity sites degradation increased rapidly as binding to low affinity sites increased. At equal levels of binding, down-regulated cells degraded insulin more rapidly than normal cells. It is concluded that: 1) insulin bound to sites of low apparent affinity is responsible for the hormone's glycogenic effect, 2) down-regulation of human hepatocytes virtually eliminates such binding and the glycogenic response and also increases the rate of degradation of insulin in relation to the amount bound to high affinity sites, 3) human cells are more sensitive than rat cells to down-regulation. It is suggested that in human cells the major effects of exposure to insulin are an inhibition of insulin internalization and an increase in the rate of degradation of that insulin which is internalized; in rat cells the major effects are a decrease in cell surface binding and an increased rate of degradation of internalized insulin.