Establishment of a human malignant meningioma cell line with amplified c-myc oncogene

A new cell line (KT21-MG1) has been established from a human malignant meningioma transplanted into nude mice. The cultured cells showed epithelial cell-like morphology and were positive immunohistochemically for vimentin as the original tumor. They have been grown continuously in vitro for more than 2 years. The population doubling time was about 24 hours at the 30th passage. The cells are capable of proliferating in soft agar medium and produced tumors in nude mice, the histologies of which were similar to the original patient-derived tumor. Analysis of cellular oncogenes showed that myc and fps were amplified approximately tenfold and threefold, respectively, in this cell line, whereas N-myc, L-myc, N-ras, K-ras, H-ras, abl, erbB2, Blym, src, raf-1, myb, and sis were not changed significantly. The amplification of myc was accompanied by an enhanced expression. Chromosome studies of cultured cells showed the monosomy of chromosome 22 that has been reported to be a specific abnormality in meningiomas.     

Amplification and overexpression of c-myc proto-oncogene correlate with the loss of glucocorticoid dependence in rodent cells transformed by human papillomavirus type 16

Transformation of baby mouse kidney epithelial cells by human papillomavirus (HPV) type 16 is dependent both upon the cooperating oncogene and on the hormonal conditions after transfection. With v-fos as the oncogene, the transformed cells require glucocorticoid hormone, such as dexamethasone, for proliferation. This requirement is lost on continued passage of cell lines, and the cells become dexamethasone independent. Steroid-independent cell lines are also produced by growth of the HPV16/v-fos cells in 17 beta-estradiol following transfection, but cell lines produced in this manner showed no subsequent requirement for estradiol or dexamethasone. Expression of the c-myc proto-oncogene was measured in dexamethasone-dependent and independent cell lines. Dexamethasone-dependent cell lines all exhibited low level c-myc expression, but this markedly increased in the cell lines that had become dexamethasone independent as a result of continued in vitro growth. The low level of c-myc expression in some early passage dexamethasone-dependent cell lines appears to be associated with rearrangement of the c-myc locus, whereas late passage dexamethasone-independent cell lines contain amplified c-myc sequences. Dexamethasone-independent cell lines derived by growth in 17 beta-estradiol showed higher levels of c-myc expression, together with higher c-myc copy number, than dexamethasone-dependent lines. Taken together, these studies indicate that the steady-state level of c-myc expression affects the continued requirement of HPV16-transformed cells for dexamethasone.     

Establishment of a peripheral T-cell lymphoma cell line showing amplification of the c-myc oncogene

A new T-cell lymphoma cell line, designated T34, was established from freshly isolated lymph node tumor cells of a patient with non-Hodgkin's diffuse large cell lymphoma. The T34 cells, as well as the parental lymphoma cells, showed mature helper/inducer immunophenotypes in that they formed spontaneous sheep erythrocyte rosettes and reacted with OKT-3 and OKT-4 monoclonal antibodies. They were negative for OKT-6, OKT-8, terminal deoxynucleotidyl transferase, WT-1, and HLA-DR antigens. Molecular analysis revealed that the T34 cells contained 8- to 16-fold amplified c-myc DNA. The same genetic change was observed in parental lymphoma cells, indicating that the c-myc amplification had occurred in vivo. There was no gross rearrangement of the c-myc DNA. The c-myc gene of the T34 cell line was actively transcribed into normal-sized c-myc mRNA. Cytogenetic analysis showed that both the T34 and the parental lymphoma cells had a near-triploid karyotype with multiple structural chromosome changes. The terminal end of the long arm of chromosome No. 8, the chromosomal locus of single-copy c-myc, was elongated (8q+ chromosome), perhaps reflecting the site of c-myc amplification. These data suggested that amplification of the c-myc oncogene played some role in progression and proliferation of this peripheral T-cell neoplasm.     

Properties of a non-tumorigenic human cervical keratinocyte cell line

A human cervical keratinocyte cell line, W12, has been initiated from a low-grade cervical lesion histologically diagnosed as CIN I. This cell line has, to date, undergone over 300 generations in vitro with an average doubling time of 30 hr: an aneuploid karyotype has developed with progressive in vitro growth. W12 contains HPV16 DNA present at approximately 100 copies and the state of the viral DNA over a number of passages has been examined. The HPV16 DNA is stably maintained at high copy number over several passages and restriction enzyme analysis together with electrophoresis of uncleaved viral DNA indicate that it is present predominantly as the episomal molecule. W12 cells exhibit a typical keratinocyte morphology and, when transplanted into the flank of the nude mouse, form an epithelial lesion with the histological features of CIN I/II.     

子宫颈病变中p16INK4A的表达以及与HPV16感染关系的研究

目的探讨p16^INK4A在各类子宫颈病变中的表达以及与HPV16感染关系的研究,为临床子宫颈病变的筛查提供经济、有效、快速的筛查方法。方法应用组织微阵列方法和免疫组化法对15例正常子宫颈、10例子宫颈尖锐湿疣、45例子宫颈上皮内瘤样病变、20例子宫颈浸润癌行p16^INK4A测定,用组织微阵列方法及原位杂交法行HPV16cDNA测定。结果随子宫颈病变的进展HPV16cDNA的表达增加（P〈0.001）。p16INK4A在正常子宫颈中无表达,而随着子宫颈病变的进展,p16^INK4A的表达率显著升高（P〈0.001）,且与HPV16有相关性。p16INK4A在子宫颈癌中的表达率为95.0。结论组织微阵列技术适用于大样本量的检测,是一种高效、快速、可比性强的分子生物学研究方法。p16^INK4A在一定程度上可反映HPV的感染程度,可作为子宫颈癌筛查的指标。p16^INK4A在子宫颈癌的发生中可能起着重要作用。     

Increased level of amplification of the c-myc oncogene in tumors induced in nude mice by a human breast carcinoma cell line

Cell line SW 613-S, derived from a human breast carcinoma, contained double minute chromosomes (DMs) but lost them progressively upon in vitro cultivation. These cells were tumorigenic in nude mice. Cell lines were derived from the tumors and were found to have a high DM content. In three such cell lines, DMs were stably maintained upon in vitro cultivation, whereas in another they were progressively lost. We found that the c-myc oncogene is amplified 5- to 10-fold in SW 613-S and 20- to 90-fold in the different cell lines derived from the tumors. At least part of the additional c-myc copies were found associated with a purified DM fraction. In cell lines which lost the DMs during in vitro passages, the level of amplification was maintained. In situ hybridization experiments indicated that this loss was compensated by the acquisition of copies of the c-myc gene integrated into a chromosome. No major rearrangement of the amplified c-myc gene was detected. The amount of c-myc messenger RNAs is roughly proportional to the level of amplification. Our results indicate that growth of SW 613-S cells as tumors in nude mice selected cells with an increased level of amplification and expression of the c-myc oncogene.     

Establishment and characterization of human signet ring cell gastric carcinoma cell lines with amplification of the c-myc oncogene.

Two unique human signet ring cell gastric carcinoma cell lines (designated HSC-39 and HSC-40A) were established in vitro from the ascites of a 54-year-old male patient. Both cell lines were biologically quite similar, grew in vitro in suspension with a population doubling time of 28-30 h, and had cytological features of mucinous epithelial tumor cells. They formed colonies in soft agar, with a cloning efficiency of 0.8-1.0%. Ultrastructurally, numerous granules were observed in the cytoplasm, suggesting secretory activity. The frequent presence of desmosome and the tight junction at the cell boundary certifies the epithelial origin of the lines. Immunocytochemistry and radioimmunoassay showed production of tumor marker antigens (carcinoembryonic antigen, CA 19-9, and sialyl-Lex-i) and gastrin in both lines. These lines were transplantable in athymic BALB/c nude mice. The histopathology of each line growing in athymic BALB/c nude mice was similar to that of the original tumor. The karyotype of the cells was highly aberrant with structural and numerical changes. The presence of numerous double minute chromosomes and loss of the 13 chromosome and Y-chromosome characterize these lines. In addition, the amplified c-myc oncogene (16-32-fold) was found in both cell lines and original ascitic tumor cells. Overexpression of the c-myc mRNA was noted. These cell lines may be a useful tool, providing both in vivo and in vitro systems for further studies of the biology and therapy of human signet ring cell (or Borrmann's type IV carcinoma) gastric carcinoma.     

Indole-3-carbinol prevents cervical cancer in human papilloma virus type 16 (HPV16) transgenic mice.

Mice that express transgenes for human papillomavirus type 16 under a keratin 14 promoter (K14-HPV16 mice) develop cervical cancer when they are given 17beta-estradiol chronically. We asked whether the antiestrogenic phytochemical indole-3-carbinol (I3C), found in cruciferous vegetables, administered at physiological doses, would prevent the cervical-vaginal cancer that is promoted in these mice by high doses of estrogen. We compared mice that were fed a control diet with those that were fed a diet supplemented with 2000 ppm I3C. In the group fed the control diet, at a dose of estradiol of 0.125 mg per 60-day release, 19 of 25 transgenic mice developed cervical-vaginal cancer within 6 months, and the remainder had dysplasia. Only 2 mice of 24 in the group fed the I3C supplemented diet developed cancer, and the remainder had dysplasia or hyperplasia. I3C reduced dysplasia in the nontransgenic mice. Similar results were obtained at a higher dose of estradiol (0.250 mg per 60-day release), and I3C helped to prevent morbidity associated with retention of fluid in the bladder that frequently occurred with the higher estradiol dose. Additionally, I3C appeared to reduce skin cancer in transgenic mice. These data indicate that I3C is a useful preventive for cervical-vaginal cancer and, possibly, other cancers with a papillomavirus component.     

In vivo amplification and rearrangement of c-myc oncogene in human breast tumors

We have studied the genomic organization of cellular myc (c-myc) proto-oncogene in 48 human primary breast tumors. Two types of alterations (amplification and rearrangement) were observed in 27 (56%) of the tumors studied. The c-myc proto-oncogene appeared to be amplified 2- to 15-fold in the DNA of 20 tumors (41%). Non-germ line c-myc-related fragments (rearrangements) of variable size were detected in 7 primary breast tumors (6 malignant, 1 benign); 4 of these tumors presented both rearrangement and amplification, and the other 3 presented rearrangement only. The majority of the tumors analyzed were invasive ductal adenocarcinomas; 58% of these showed c-myc locus genetic alterations. Although the c-myc alterations described here do not appear to correlate with the aggressive behavior of primary breast tumors, they seem to be associated with development of breast carcinoma.     

人类子宫颈癌基因、p16及人乳头瘤病毒16／18在子宫颈癌中表达的意义

 目的　研究人类子宫颈癌基因（HCCR）、p16在子宫颈上皮内瘤变（CIN）及子宫颈癌中的表达, 探讨其与人乳头瘤病毒（HPV）16／18感染的相关性及其临床意义。方法　采用免疫组织化学SP法检测28例慢性子宫颈炎,62例CINⅠ~Ⅱ级、49例CINⅢ级及52例子宫颈癌组织中p16、HCCR蛋白的表达,并采用原位杂交法检测其HPV16／18的表达。结果　HCCR在慢性子宫颈炎、CINⅠ~Ⅱ级、CINⅢ级和子宫颈癌组的阳性表达率分别为3.57 %（1／28）、35.48 %（22／62）、61.22 %（30／49）和88.46 %（46／52）,各组间两两比较,差异有统计学意义（P＜0.05）。p16蛋白的阳性表达率从慢性子宫颈炎、CINⅠ~Ⅱ级、CIN Ⅲ级到子宫颈癌组逐渐增加,分别为7.14 %（2／28）、33.87 %（21／62）、65.30 %（32／49）和92.31 %（48／52）,各组间两两比较差异均有统计学意义（P＜0.05）;HPV16／18在慢性子宫颈炎、CINⅠ~Ⅱ级、CINⅢ级和子宫颈癌组的阳性率分别为10.71 %（3／28）、40.32 %（25／62）、69.39 %（34／49）和84.62 %（44／52）,除子宫颈癌与CINⅢ级比较差异无统计学意义外（P＝0.115）,其余组间两两比较,差异有统计学意义（P＜0.01）。p16、HCCR的表达与HPV16／18感染呈正相关（P＜0.01）。结论　HPV感染可能通过影响p16及HCCR蛋白的过表达与子宫颈癌及其癌前病变的发生、发展密切相关,三者联合检测对子宫颈癌及癌前病变的筛查和预防具有重要意义。     

Proliferation of the human colon carcinoma cell line HT29: autocrine growth and deregulated expression of the c-myc oncogene

Human colon adenocarcinoma cell (line HT29) are able to proliferate in a defined (serum-free) medium containing no added growth factors; in such conditions, their doubling time is 3 to 4 days (on serum-coated dishes) or 2 to 3 days (on an autologous extracellular matrix) compared with 1 day in the presence of fetal calf serum. In the presence of suramin, a polyanion disrupting the binding of growth factors to their receptors, the incorporation of [3H]thymidine in serum-free cultures is reduced (27.0 +/- 2.9% of control after 3 days of culture), suggesting involvement of autocrine growth factors in the autonomous proliferation of the cells. The expression of the proliferation-related oncogene c-myc was examined during various stages of growth and differentiation of the HT29 cells. The cellular contents of c-myc mRNA were similar in all experimental conditions studied: exponential phase; stationary phase; nondifferentiated as well as differentiated cells (by glucose deprivation); and also in serum-free medium containing or not suramin. An approximately 2-fold increase in the level of c-myc mRNA was observed in cells cultured for 3 days in suramin-containing medium and then incubated during 3 h in the absence of suramin (with or without 10% fetal calf serum). Southern blot analysis of the genomic DNA of HT29 cells did not reveal any rearrangement within the region containing the c-myc gene and the flanking sequences (approximately five kilobases upstream and approximately three kilobases downstream). The c-myc locus was weakly amplified (four to six copies per cell). These results indicate that the c-myc gene expression in HT29 cells is deregulated and does not require growth factor stimulation. The deregulation of the c-myc gene may be related to the reduced growth factor requirement of the HT29 cell line.     

Presence of human papilloma virus types 16 and 18 in genital warts and cervical neoplasias

Certain types of human papilloma viruses (HPV) are associated with human genital proliferative diseases, and among them HPV16 and HPV18 seem to play an important role in the occurrence of cervical cancer. We used restriction enzyme analysis and molecular hybridization, in order to investigate the type of viral infection and the physical state of viral DNA in gynecological benign, pre-malignant and malignant lesions. HPV6/11 specific sequences could only be detected as episomes and this in benign lesions classified as condylomata acuminata. On the other hand, HPV16 and HPV18 sequences were detected in non-malignant lesions such as flat condylomata (7 out of 14 cases), pre-malignant lesions including cervical intra-epithelial neoplasias (10 out of 20 cases), and most frequently in cervical invasive cancers (21 out of 27 cases). In a large number of virus-positive cases, HPV16 and HPV18 could only be discerned in forms consistent with the existence of episomes and/or randomly integrated head-to-tail oligomers. However, some invasive carcinomas and cervical intra-epithelial neoplasias contained, in addition, clonal outgrowths with detectable virus-cellular junction fragments of the integrated viral genomes. In the light of these data, monitoring the type of viral infection proves to be an important adjunct to histological analysis when assessing those patients affected by condyloma or cervical intra-epithelial neoplasia who are at risk for developing invasive carcinoma.     

人乳头瘤病毒水平与宫颈局部免疫功能对高危型人乳头瘤病毒感染宫颈癌患者的影响

目的 探讨人乳头瘤病毒(HPV)水平与宫颈局部免疫功能对高危型HPV感染宫颈癌患者的影响.方法 收集76例宫颈癌患者(宫颈癌组)、40例慢性宫颈炎患者(慢性宫颈炎组)和40例宫颈上皮内瘤变患者(宫颈上皮内瘤变组)临床资料.检测并比较3组患者HPV水平和免疫功能指标[CD4+、CD8+、CD4+/CD8+、CD56+和调节性T细胞(Treg)]水平;比较有无淋巴结转移宫颈癌患者HPV水平和免疫功能指标水平;比较死亡和生存宫颈癌患者HPV水平和免疫功能指标水平.应用Pearson相关性分析法分析HPV水平与各免疫功能指标的相关性.结果 宫颈癌组患者HPV、CD8+和Treg水平最高,宫颈上皮内瘤变组患者次之,慢性宫颈炎组患者最低,差异均有统计学意义(P﹤0.05);宫颈癌组患者CD4+、CD4+/CD8+、CD56+水平最低,宫颈上皮内瘤变组患者次之,慢性宫颈炎组患者最高,差异均有统计学意义(P﹤0.05).淋巴结转移和死亡宫颈癌患者CD4+、CD4+/CD8+、CD56+水平均低于无淋巴结转移和生存患者,HPV、CD8+、Treg水平均高于无淋巴结转移和生存患者,差异均有统计学意义(P﹤0.05).CD4+、CD4+/CD8+和CD56+与HPV水平均呈负相关(r=-0.48、-0.35、-0.40,P﹤0.05),CD8+、Treg与HPV水平均呈正相关(r=0.45、0.26,P﹤0.05).结论 高危型HPV感染宫颈癌患者HPV水平呈异常高表达且患者宫颈局部免疫功能明显降低,是导致患者持续感染高危型HPV和加重宫颈病变的重要原因.     

Enhancement of human papilloma virus type 16 E7 specific T cell responses by local invasive procedures in patients with (pre)malignant cervical neoplasia

It has been suggested that local invasive procedures may alter the natural course of (pre)malignant cervical disease. This could be due to partial excision of the lesions, or via induction of cellular immunity against human papillomavirus (HPV) by the local invasive procedures. We studied the influence of local invasive procedures on HPV-16 E7 specific immune responses in patients with different grades of cervical intra-epithelial neoplasia (CIN) and different stages of cervical cancer. Blood was obtained at intake and after invasive procedures from patients with CIN or cervical cancer. Antigen specific T-cell responses were measured by IFN-gamma ELISPOT analysis, after stimulation with recombinant HPV-16 E7 protein. As expected, HPV-16 E7 specific IFN-gamma T cell responses were more frequent in HPV-16 DNA positive patients compared with that in HPV-16 DNA negative patients (39/50 vs. 16/36, (p=0.006, chi2 test). After invasive procedures, a small number of HPV-16 DNA positive CIN patients, but a considerable proportion of HPV-16 DNA positive cervical cancer patients, showed an enhancement of T cell responses against HPV-16 E7. Induction of T cell reactivity was most pronounced in cervical cancer patients who had undergone previous invasive procedures. Both CD4+ and CD8+ T cells showed E7 specific IFN-gamma production upon in-vitro stimulation. Our study shows that invasive procedures may enhance HPV-specific cell-mediated immunity in a considerable number of patients with cervical cancer, but in only a minority of CIN patients. Our data indicate that invasive procedures should be considered as possible confounding factors when analyzing the effectiveness of therapeutic immunization studies, especially, when induction of HPV-specific immune responses is used as intermediate end-point.     

Favorable clinical outcome of cervical cancers infected with human papilloma virus type 58 and related types

To determine whether the status of human-papillomavirus (HPV) infection affects the clinical outcome of cervical carcinoma (CC), HPV genotype was prospectively determined in 94 consecutive CC cases subsequently followed for a median duration of 37.5 months. With a consensus PCR-RFLP method of HPV genotyping, 81 (86.2%) cancers were positive for HPV DNA. They were classified, according to the phylogenic similarities, into HPV-16-related (type 16, n = 45; type 31, n = 2), HPV-58-related (type 58, n = 17; type 33, n = 3; type 52, n = 2) and HPV-18-related (type 18, n = 8; type 68, n = 1) groups, and analyzed in relation to clinical outcome. The following results were observed: (i) Type-58-related HPVs were more prevalent in the old age (older than the median age of 52) group than in the young age group (41% vs. 14.6%, p = 0.045); (ii) 63% (5/8) of patients with advanced stages (III and IV) were HPV-negative, a figure much higher than that (9.3%, 8/84) of patients with early stages (stage I and II) (p = 0.002); (iii) the occurrence of adenocarcinoma or adenosquamous carcinoma was higher in the HPV-18-related group (50%) than in the HPV-16-related (33.3%) or the HPV-58-related (16.7%) groups (p = 0.024); (iv) the status of lymph-node metastasis and tumor grade did not correlate with HPV status; (v) 5-year survival rates were 90.2%, 80% and 74% for HPV-58-, HPV-16- and HPV-18-related groups, respectively (p = 0.03, after adjustment for tumor stage); (vi) in comparison with the HPV-16-related group, the relative risk of death in the HPV-58- and the HPV-18-related groups were 0.32 [95% CI, 0.07-1.49] and 1.87 [0.36-14.9] respectively. HPV genotype appears to affect the clinical behavior and outcome of cervical cancer. HPV-58-related types are prevalent in the older population, and appear to confer a favorable prognosis. Int. J. Cancer (Pred. Oncol.) 84:553-557, 1999.     

Genotypes of human papilloma virus in Sudanese women with cervical pathology

Background  

Knowledge of the distribution of human papillomavirus (HPV) genotypes among women with cervical lesion and in invasive cervical cancer is crucial to guide the introduction of prophylactic vaccines. There is no published data concerning HPV and cervical abnormalities in Sudan. This study aimed to define the prevalence of HPV and its subtypes in the cervical smears of women presenting with gynecological complains at Omdurman Military Hospital, Sudan.     

High-risk human papilloma virus and cervical abnormalities in HIV-infected women with normal cervical cytology

Background

The prevalence of High-Risk Human papilloma virus (HR-HPV), a necessary cause of invasive cervical cancer (ICC) is relatively high in HIV infected women. Gaps exist in our knowledge of the optimal approaches for managing women who have HR-HPV with normal cervical cytology (NCC) particularly in settings of HIV infection.

Methods

Between May 2012 and June 2013 we conducted a colposcopic assessment of HIV-infected women with prior (NCC) and known HR-HPV status to compare cervical abnormalities in women with and without HR-HPV. Colposcopic examinations were done at the Operation Stop Cervical Cancer (OSCC) unit of the Jos University Teaching Hospital (JUTH), Jos, Nigeria. Abnormal colposcopic finding (ACF) was defined as areas of aceto-white epithelium involving the squamo-coulumnar junction, areas of punctation, mosaic pattern or atypical vessels. We compared proportions of ACF as well as histologic grades of cervical intra-epithelial neoplasia (CIN) in women with or without HR-HPV. Statistical analysis was done on STATA.

Results

We conducted colposcopic examinations in 78 out of 89 (86.5%) eligible women. The mean age of the cohort was 32.4 years (SD ±4.6) with a median 32 years (IQR 29–36). After a mean follow up time of 20.1 months from the initial cervical pap cytology and HR-HPV testing, we found 12 of 78 (15.4%) women with ACF. The odds for an ACF was statistically higher [OR = 4.0 (95% CI: 1.1-14.7)] in women with HR-HPV compared to those without. Of the twelve women with ACF, subsequent histologic examination of colposcopically directed cervical biopsies confirmed CIN 1 in 4 cases (33.3%), CIN 2 in 1 case (8.3%), CIN 3 in 2 cases (16.7%), carcinoma-in-situ (CIS) in 2 cases (16.7%), and normal cervix in 3 (25.0%). Overall, the proportion of women detected with any grade of CIN was 11.5% (9/78) and 6.4% (5/78) were CIN 2 or greater lesion (CIN2+).

Conclusion

HIV-infected women with NCC and HR-HPV had a four-fold higher likelihood for an ACF. The practice of early colposcopic examination of HIV-infected women with prior NCC and HR-HPV may increase early detection of higher grade CIN and CIS cancer stages in our setting.