P53 protein expression and DNA ploidy in common epithelial tumors of the ovary

OBJECTIVE: To investigate p53 protein expression and DNA content in imprints from surgical biopsies of common epithelial tumors of the ovary. STUDY DESIGN: The study was based on 60 cases of epithelial tumors of the ovary (15 benign, 3 border-line and 42 malignant). For the demonstration of p53 protein, immunocytochemical staining with the avidin-extravidin technique was performed using monoclonal antibody p53 DO-7. DNA content was measured by image cytometry after Feulgen staining. RESULTS: There was a strong correlation between p53 expression and aneuploidy, with the difference between diploid and aneuploid tumors statistically significant (P < .001). A correlation was found between DNA ploidy, histologic grade and clinical stage (P < .001 and P < .05), respectively. There was no correlation between DNA ploidy and histologic type (P = .89). No correlation was observed between p53 protein expression and grade or clinical stage of the tumors. Nevertheless, a correlation of p53 expression between early (I, II) and advanced stages (III, IV) (P < .05) was observed. All benign and borderline tumors were diploid and did not express p53 protein. CONCLUSION: The results of the present study and the data in the literature stress the value of p53 expression and DNA ploidy in assessing the malignant potential of common epithelial ovarian cancers. However, the clinical application of these data requires further study.     

Apoptosis and cellular proliferation in human epidermal squamous cell neoplasia

The structural changes in outpatient medical care in eastern Germany took place by far-reaching winding up of the outpatient departments. At the same time, many doctors from these institutions set up a practice. These changes also concerned the medical care by family doctors. It is assumed that as a result of these partly extensive changes there are also changes in the accessibility of the family doctor by outpatients. We will describe in this paper the level attained in Dresden, capital of Saxony in 1995 and 1996, from the point of view of the inhabitants. Existing problems are shown. In this connection, random samples of inhabitants of Dresden were interviewed by postal survey with questions on this subject in 1995 and 1996.     

DNA ploidy and expression of p53 and C-erbB-2 in extramammary Paget''s disease of the vulva

We report a case of total pulse alternans in a patient with paroxysmal ectopic atrial tachycardia and echocardiographic findings obtained before and after radiofrequency catheter ablation (RFCA) that terminated the arrhythmia. The patient was a 27-year-old man with history of paroxysmal palpitations with worsening episodic dizziness, chest tightness, and dyspnea. Electrocardiography (ECG) showed atrial tachycardia at 160 to 170 beats/min while the simultaneous pulse was in the 80s beats/min. Echocardiogram showed that aortic and mitral valves opened with alternating excursions and outflow velocities. Furthermore, despite similar ventricular wall thickening during systole of consecutive cardiac cycles, there was alternating mitral valve opening during diastole of the same cycles, providing direct evidence that ineffective diastolic filling and mitral valve opening may play a role in the pathogenesis of pulse alternans. Repeat ECG and echocardiography after the successful RFCA showed normal sinus rhythm and normal opening excursion and the velocity across the aortic and mitral valves.     

DNA ploidy and MYC DNA amplification in ovarian carcinomas. Correlation with p53 and bcl-2 expression, proliferative activity and prognosis

There is increasing evidence that DNA ploidy is a prognostic factor in ovarian carcinomas, but it is uncertain whether MYC DNA amplification is an epiphenomenon of DNA nondiploidy or a distinct biological change with an impact on the clinical course of the disease. To clarify these issues we analysed DNA ploidy by flow and image cytometry and MYC copy number by polymerase chain reaction in archival material from ovarian carcinomas with known follow up. The results were compared with proliferative activity (Ki67 index) and p53 and bcl-2 expression. DNA cytometry revealed nondiploidy in 84 of 144 cases (58.3%). Nondiploidy was statistically significantly correlated with histological tumour type, histological grade, Ki67 index > 10%, FIGO stage, presence of residual tumour after debulking surgery and adverse postoperative outcome. Furthermore, DNA nondiploidy was associated with p53 accumulation. We found that 84.9% of the p53-positive cases were nondiploid. This points to the paramount importance of wild type p53 for the maintenance of genome integrity in this tumour type. MYC DNA amplification was seen in 33.8% (26/77 cases) of ovarian carcinoma. There was no correlation between MYC DNA amplification and histological tumour type, histological grade, FIGO stage, DNA ploidy, proliferative activity or prognosis. However, when p53 and bcl-2 expression was taken into account, a statistically significant correlation between gene alteration or expression patterns and histological tumour type was revealed. The group of mucinous carcinomas demonstrated both MYC DNA amplification and strong bcl-2 expression in 50% and contained the largest fraction of cases without aberration (37.5%). Endometrioid carcinomas were characterized by strong bcl-2 expression in 85%, whereas serous and undifferentiated carcinomas predominantly exhibited p53 alterations, frequently accompanied by bcl-2 overexpression or MYC DNA amplification. Thus, in interaction with other genes MYC DNA amplification may play a role in the determination of the varying differentiation patterns of ovarian carcinomas.     

Bcl-2 but not Bax expression is associated with apoptosis in normal and transformed squamous epithelium

Aberrant regulation of apoptosis may contribute to tumorigenesis. Relative levels of apoptosis regulatory proteins, such as Bcl-2 and Bax as well as interactions of these proteins with other gene products, may contribute to the rate of apoptosis in neoplasia. We examined Bcl-2 expression in 104 squamous cell carcinomas of the head and neck, as well as histologically normal mucosa several centimeters away from the tumor, and in control normal mucosa from patients without cancer. Immunohistochemistry and immunoblotting demonstrated Bcl-2 expression in 30% (31 of 104) of squamous cell carcinoma, with an increase in Bcl-2 protein levels compared with control normal mucosa from noncancer patients. Bcl-2-positive tumors demonstrated a 5-fold decrease in the number of apoptotic cells compared with Bcl-2-negative tumors. Bcl-2 protein expression was associated with poorly differentiated tumor grade but was not correlated with Bax expression or patient survival. These findings demonstrate that Bcl-2 contributes to apoptosis in normal and transformed squamous epithelium.     

Aberrant FHIT transcripts in cancerous and corresponding non-cancerous lesions of the digestive tract

We lengthened seven first metatarsals in four patients with short great toes by callus distraction using an external fixator. Good clinical and cosmetic results were obtained. Bone lengthening is effective in patients with short great toes not only for cosmesis, but also to relieve pain and callosities on the plantar aspect of the second and third metatarsal heads. Excessive lengthening of the first metatarsal resulted in limitation of the range of movement of the metatarsophalangeal joint of the great toe. To prevent this the amount of lengthening should not exceed 40% of the preoperative length of the metatarsal.     

Comparative image cytometric DNA ploidy of liver cell dysplasia and hepatocellular carcinoma

Large-cell liver cell dysplasia (LCD), suggested to be a preneoplastic change that progresses to hepatocellular carcinoma (HCC), has a reported frequency of DNA aneuploidy by flow cytometry intermediate between that of nonneoplastic liver (0%) and HCC (80%). We assessed DNA ploidy by image cytometry of Feulgen-stained 5-microns sections of 30 livers with LCD and of 60 HCCs (29 with LCD in adjacent nonmalignant liver). All 30 LCDs were aneuploid, 27 (90%) of which were multiploid--11 (41%) with hyperdiploid and hypertetraploid peaks. Forty-eight (80%) HCCs were aneuploid; in nine of 20 (42%) with a hyperdiploid peak, a hyperdiploid peak was also present in the LCDs, but in none was there less than 0.24 between DNA indices. Besides the 12 (20%) diploid HCCs, a diploid peak was present in four heterogenous, three multiploid, and six HCCs with two phenotypes and two genotypes, one of which was diploid. One aneuploid/hyperdiploid peak in each of 22 nonneoplastic and 24 cirrhotic livers did not have a corresponding LCD or HCC aneuploid peak. These data do not suggest that dysplastic hepatocytes form a single mutant clone that progresses to HCC.     

DNA quantification and ploidy patterns in human adrenocortical neoplasms

The determination of DNA ploidy and the study of the cell cycle in adrenocortical tumoral cells could help in the distinction between benign and malignant lesions and also in the prediction of the biological behaviour of these tumors. We analysed 32 cases of adrenal tissue (8 normal adrenals--N, 12 benign adenomas--A and 12 carcinomas--C). DNA was quantified by image analysis of Feulgen stained sections (Ahrens System) employing ACAS3 software. The DNA content was considered to be diploid in 70% of the N and in 67% of the A groups and in none of C. In this latter group nearly 90% were triploid or tetraploid while this did not occur in any of the A cases. The percentage of cases with a 5c-exceeding rate (5cER) above 5% was nil in the N and A groups and 100% in C. In what concerns the distribution in the cell cycle we found a very distinctive pattern between the groups as the percentage of cases in which the S-phase fraction exceeded 33% was nil in the normals, 8% in A and 83% in the C cases. In conclusion, there was a good correlation between the analysed parameters and the clinically defined groups of adrenal cortex tumors.     

Detection of p53 protein overexpression and DNA ploidy analysis in colon cancer

BACKGROUND/AIMS: This study was designed to demonstrate the accumulation of the mutant p53 protein in human neoplasms. The correlation of flow cytometric DNA ploidy pattern with p53 expression using the immunoblotting technique was also investigated. METHODOLOGY: In this study, the occurrence of p53 overexpression was analyzed in 34 cases of adenocarcinoma of the colon by western immunoblotting technique, using an anti-human p53 monoclonal antibody (Do-7). The nuclear protein extract from human colon tumor specimens was immunoblotted relative to protein standards of known molecular weight. Flow cytometric analysis was used to study the DNA ploidy pattern of the tumor cells. RESULTS: Monoclonal antibody p53-Do 7 detected a single band of 53 KDa in 70.5% (24 of 34) of the tumor specimens examined. Whereas, no bands were detected in the normal colon mucosa. The relation between p53 overexpression and the clinicopathological variable (Dukes' staging) was studied and no significant difference in p53 overexpression between Dukes' stages B and C was found. Flow cytometric analysis revealed a higher incidence of DNA aneuploidy in 75% (15 of 20) of p53 positive cases compared with 64.3% (9 of 14) in the diploid tumors. CONCLUSION: The immunoblotting technique can successfully detect the mutant p53 and is therefore expected to provide valuable information on the role of p53 in the process of carcinogenesis.     

Assessment of dysplasia, mucosal mucins, p53 protein expression, and DNA content in ulcerative colitis patients with colectomy and ileorectal anastomosis

BACKGROUND: Patients with ileorectal anastomosis after colectomy for ulcerative colitis remain at risk of developing rectal malignancy. Detection of mucosal dysplasia has been used for regular screening but is difficult in inflammatory mucosa, prompting the search for complementary markers. METHODS: This prospective study aimed to assess the prevalence of dysplasia, the predominance of sialomucin, DNA aneuploidy, and p53 overexpression as possible predictors of colorectal tumourigenesis, in the rectal mucosa of an unselected group of 27 patients with ileorectal anastomosis performed for ulcerative colitis. Patients had neither neoplastic nor dysplastic lesions on the colectomy specimen and the retained rectum at the time of surgery. One biopsy specimen of each lateral rectal wall was studied, using routine histology, mucin histochemistry, DNA flow cytometry, and the streptavidin-biotin complex method with D07 monoclonal antibodies directed towards the p53 protein. RESULTS: Seventeen, seven, and three patients showed inflammatory lesions of inactive, moderate, and severe active colitis, respectively. Dysplasia, sialomucin predominance, DNA aneuploidy, and p53 overexpression were not detected. CONCLUSIONS: The risk of malignant transformation of the rectal mucosa after ileorectal anastomosis seemed to be low in this ulcerative colitis group without high-grade dysplasia or carcinoma in the previous colectomy specimen, carefully followed up endoscopically and histologically. It remains to be evaluated which of the methods studied above will optimize the histopathologic surveillance of the rectal mucosa of ulcerative colitis patients with ileorectal anastomosis.     

Cell proliferation and ploidy of human solid tumours: methodological experience with in vivo bromodeoxyuridine and DNA flow cytometry

The divalent cation selective ionophores A23187 and ionomycin were compared for their effects on the Ca2+ contents, nucleotide contents, and protein synthetic rates of several types of cultured cells. Both ionophores reduced amino acid incorporation by approximately 85% at low concentrations (50-300 nmol/L) in cultured mammalian cells without reducing ATP or GTP contents. At these concentrations A23187 and ionomycin each promoted substantial Ca2+ efflux, whereas at higher concentrations a large influx of the cation was observed. Ca2+ influx occurred at lower ionophore concentrations and to greater extents in C6 glioma and P3X63Ag8 myeloma than in GH3 pituitary cells. The ATP and GTP contents of the cells and their ability to adhere to growth surfaces declined sharply at ionophore concentrations producing increased Ca2+ influx. Prominent reductions of nucleotide contents occurred in EGTA-containing media that were further accentuated by extracellular Ca2+. Ionomycin produced more Ca2+ influx and nucleotide decline than comparable concentrations of A23187. The inhibition of amino acid incorporation and mobilization of cell-associated Ca2+ by ionomycin were readily reversed in GH3 cells by fatty acid-free bovine serum albumin, whereas the effects of A23187 were only partially reversed. Amino acid incorporation was further suppressed by ionophore concentrations depleting nucleotide contents. Mitochondrial uncouplers potentiated Ca2+ accumulation in response to both ionophores. At cytotoxic concentrations Lubrol PX abolished protein synthesis but did not cause Ca2+ influx. Nucleotide depletion at high ionophore concentrations is proposed to result from increased plasmalemmal Ca2+-ATPase activity and dissipation of mitochondrial proton gradients and to cause intracellular Ca2+ accumulation. Increased Ca2+ contents in response to Ca2+ ionophores are proposed as an indicator of ionophore-induced cytotoxicity.     

Detection of human papillomavirus DNA sequences in oral squamous cell carcinomas and their relation to p53 and proliferating cell nuclear antigen expression

BACKGROUND: The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site. METHODS: Seventy-seven oral mucosal SCCs were examined for the presence of HPV DNAs by polymerase chain reaction and dot blot hybridization. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and p53 was performed and single strand conformation polymorphism analysis for p53 was undertaken. In situ hybridization detection of HPV-16 DNA also was performed. RESULTS: Human papillomavirus-16 DNA was detected in 23 cases of oral SCC and both HPV-16 and HPV-18 DNA were detected in one case of tongue SCC. Human papillomavirus DNAs were detected of 11 of 33 tongue, 4 of 15 gingival, 2 of 4 palate, 2 of 5 buccal mucosa, 3 of 7 maxillary sinus, and 2 of 11 the floor of the mouth SCCs. None were detected in SCCs of the retromolar region (0/2). Immunohistochemical examination for p53 was performed in 26 cases of oral SCC and the accumulation of p53 protein was observed in 6 cases (i.e., in 4 of 17 HPV DNA-negative cases and in 2 of 9 HPV DNA-positive cases). Single strand conformation polymorphism analysis confirmed gene mutations in all 6 cases. Human papillomavirus-16 DNA was predominantly identified in cancer cells that showed a morphologic resemblance to basal cells and its hybridized signal in keratinized cells was reduced by in situ hybridization detection. Immunohistochemical detection of PCNA revealed its cooccurrence with HPV-16 DNA in cancer cells. CONCLUSIONS: These results suggest that HPV-16 DNA sequences may have the capability to maintain the proliferative state of epithelial cells, and may contribute to the production of malignant phenotypes.     

Relationships among tenascin expression, DNA ploidy patterns, and multidrug resistance gene product (P-glycoprotein) in human colon carcinoma

Relationships among tenascin expression, DNA ploidy, and P-glycoprotein were examined in 81 primary human colon cancers and 61 metastatic lymph nodes. First, the DNA ploidy patterns of colon cancerous tissue surrounded (TN+) and not surrounded (TN-) by tenascin immunoreactivity were investigated. Then the expression of P-glycoprotein, one of two multidrug resistance gene products, was examined in TN+ and TN- colon cancer tissues by immunohistochemistry. Aneuploid DNA patterns were observed at high frequency in TN- colon cancer tissues (37/61) and metastatic lymph nodes (44/52). In contrast, diploid DNA patterns were observed predominantly in TN+ colon cancer tissues (50/56). Although P-glycoprotein expression was observed in primary TN+ and TN- colon cancer (9/81), the level of P-glycoprotein expression was not correlated with DNA aneuploidy in TN- colon cancer tissues. Overall, reduced tenascin expression was correlated well with DNA aneuploidy, but no significant correlation was found between DNA aneuploidy and P-glycoprotein appearing when cancer cells become resistant to several anti-cancer drugs. Thus, tenascin may play an important role in preventing colon cancer cells from invading surrounding tissues.     

Distinct P-cadherin expression in cultured normal human keratinocytes and squamous cell carcinoma cell lines

A thermal threshold measurer (TTM) apparatus was developed and tested in 12 dry, nonpregnant, culled cows with the purpose of measuring the thermal nociceptive threshold and of finding the response to morphine sulphate dosages. The cows received a cumulative dose (from 0.00 to 0.40 mg/kg BW) of morphine sulphate through a catheter in the jugular vein. The interval between doses was 20 min, and a nociceptive test was performed 15 min after each injection. The TTM device consisted of a 60 W halogen bulb mounted in a 15 cm PVC tube, with a 0.6 s response time probe attached to its end, connected to a thermocouple. The probe measured the response temperature on the skin over the middle phalanges on the dorsum of the forefoot. The radiating heat stimulus from the bulb was instantaneously terminated with the foot-lift response of the tested animal. The nociceptive response to the 0.00 mg/kg dose was considered the baseline and subsequent measurements were expressed in difference from it. Data were evaluated in a regression analysis using the GLM procedure. A significant elevation (P < 0.0001) in the nociceptive threshold of the cows with cumulative dosing of morphine sulphate was noticed. A high variability (P < 0.0001) in the response among animals was also detected, suggesting that a 2-step dose of morphine sulphate is necessary to achieve a certain degree of induced analgesia in all cows. The nociceptive assay described, using the TTM device, was able to detect an elevation of the thermal threshold of cows due to morphine sulphate induced analgesia. An increase in locomotory behaviour or other side effects due to morphine sulphate were not noticed.     

p53 expression, DNA content and cell proliferation in primary and synchronous metastatic lesions from ovarian surface epithelial-stromal tumours

The aim of this study was to investigate biological heterogeneity between primary and metastatic ovarian cancer lesions from individual patients as a means of elucidating steps in clinical progression. Cancer tissue from 61 untreated patients with ovarian surface epithelial-stromal tumours was examined. p53 expression detected immunocytochemically by the PAb1801 antibody, DNA content evaluated by flow cytometry, and cell proliferation evaluated as the [3H]thymidine labelling index were investigated in primary tumours and corresponding synchronous metastases. The frequency of p53 positivity was similar in primary (62%) and metastatic (66%) sites, with an agreement between the two lesions from the same patient in 97% of the cases. Similarly, aneuploidy frequency (80%) and DNA indices were superimposable in primary and metastatic lesions from the same patient, with a 94% agreement. The frequency of aneuploidy was higher in p53-positive than in p53-negative lesions. An overall poor agreement (rs = 0.44) was observed for proliferative activity of primary and metastatic lesions, due to a heterogeneous profile in omental with respect to primary tumours, which was mainly evident in p53-positive cancers. Conversely, cell proliferation of peritoneal, abdominal and pelvic lesions was qualitatively similar to that of the primary tumour in 88% of patients.     

Relationship between expression of P21ras and cellular DNA in pleomorphic adenoma of lacrimal gland

BACKGROUND: The pleomorphic adenoma is the most frequent tumor of the human lacrimal gland comprising about 50% of the epithelial tumors of this organ. Although being benign, local recurrences can occur when the first removal was incomplete and malignant transformation is also not in frequent. It is well known that many sorts of cellular oncogene products are involved in the initiation, promotion and progression of the human neoplasm. Our purpose was to know whether there is abnormal expression of P21ras in pleomorphic adenoma. METHODS: We have undertaken a study of the expression of P21ras in 5 normal tissues and 32 pleomorphic adenoma of lacrimal gland by immunohistochemical means using the monoclone antibody F-132-62 and the nuclear DNA content in the tumor was assayed by image analysis technique. RESULTS: Normal tissues of lacrimal gland were negative, 12 tumors were stained positively with the antibody. The DNA content of 14 cases of tumor was increased. Their DNA ploidy distribution pattern showed two or several peaks. Good correlation has been found between the expression of P21ras and DNA ploidy distribution pattern, the DNA ploidy distribution pattern of tumor which expressed p21ras showed mainly two or several peaks. P< 0.05. CONCLUSIONS: The result of our studies may suggest that there are increased expression of p21ras in pleomorphic adenoma and the expression of p21ras is related to the promotion and progression of pleomorphic adenoma of lacrimal gland.     

Relationships between p53 induction, cell cycle arrest and survival of normal human fibroblasts following DNA damage

It is now well established that in response to genotoxic stresses mammalian cells show an increased p53 protein levels and undergo cell cycle arrest at G1/S and G2/M checkpoints. But, the consequences of these cell cycle arrests on cell survival are not yet elucidated. In this study, we have analysed the relationships between p53 protein induction, cell cycle arrest and cell survival following exposure of normal human fibroblasts (NHFs) to various genotoxic agents such as cisplatin, UV radiation and gamma radiation. p53 protein accumulation and G2/M arrest arose at the same time following exposure to DNA damaging agents, suggesting that p53 is responsible for the G2/M block. However, following inhibition of p53 induction by an antisense oligonucleotide, this G2/M arrest is even more important and correlates with an enhanced sensitivity of NHFs to UV radiation. In addition, there appears to be a threshold in the response of NHFs to DNA damaging agents, p53 induction and cell cycle arrest being observed only with lethal UV doses. We show that: 1) there appears to be a threshold in the cellular response to genotoxic agents, below which neither p53 induction, nor cell cycle arrest, nor cell survival alteration occur and beyond which p53 induction is accompanied by cell cycle arrest and decreased cell survival; 2) although there is a tight temporal relationship, the onset of which depends of the DNA damaging agent used, between the start of p53 induction and the occurrence of G2/M arrest, this latter is independent of p53; 3) p53 inhibition enhances NHFs' sensitivity to DNA damaging agents, the extent of the G2/M arrest correlating with decreased cell survival. Finally, the lack of obligatory correlation between p53 inactivation, apoptosis and radio- or chemoresistance is discussed.     

Hydroxyurea synchronization of cellular proliferation in the esophageal epithelium of mice with tumors

The synchronizing effect of hydroxyurea on the passage of the esophagus epithelia cells through the stage of the DNA synthesis and mitosis was investigated in albino mice bearing sarcoma. Dinrnal variations in the number of epithelial cells at these stages of the mitotic cycle were taken into consideration. Two mutually complementary indices for the measurement of the shifts in the cell population and the rate of the synchronization are used. Two cell groups which passed the S-phase and mitosis synchronously were found in artificial synchronization in the esophageal epithelium. The synchronization indices characterizing the first group were lower than control. In the second group the cell count during the DNA synthesis was double as compared with the number of cells synthesizing DNA in natural synchronization; however, the rate of the changes in the synchronism was the same in the experimental and control groups.     

Fibroblast growth factor receptor expression reflects cellular differentiation in human oral squamous carcinoma cell lines

This study examined the expression of fibroblast growth factor receptor 2 (FGFR 2) splice variants, IIIb and IIIc, in normal and malignant human oral keratinocytes and in normal oral fibroblasts by RT-PCR using both exon-specific primers and primers common to both FGFR 2 isoforms. Fibroblasts expressed exclusively FGFR 2/IIIc whilst the normal and malignant keratinocytes co-expressed FGFR 2/IIIb and FGFR 2/IIIc. Well-differentiated keratinocytes expressed proportionally more FGFR 2/IIIb than IIIc whereas the poorly-differentiated cells expressed more FGFR 2/IIIc than IIIb. The normal and malignant keratinocytes, but not fibroblasts, expressed an additional amplification product, which consisted of both IIIb and IIIc of FGFR 2 joined by an extra base pair and with the intronic sequence removed. The results indicate that the expression of FGFR 2 isoforms reflects the degree of cellular differentiation in normal and malignant human oral keratinocytes and that receptor complexes of FGFR 2/IIIb and IIIc may regulate ligand-receptor interactions.