Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen.

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.

Human sperm chromosome analysis--study on human sperm chromosome mutagenesis induced by carbon disulfide.

The aim of this study was to investigate the effect CS2 of on human sperm chromosomal aberration. The human sperm/hamster egg fusion technique was used to analyze 203 human sperm chromosome complement from 9 healthy volunteers. The incidence of numerical aberration was 1.0% and that of structural chromosome aberration was 5.9% and total abnormalities was 6.9%. Structural aberrations consisted of breaks, deletions, centric rings, fragments, and chromatid exchange. The results from high concentration group (10 mumol.L-1 CS2) showed that the incidence of chromosomal aberration rate was significantly higher than that of the control group. The results indicate that high concentration of CS2 might directly cause mutagenesis of the germ cell.

Photoanthropometric study of craniofacial traits of individuals with Prader-Willi syndrome.

A photoanthropometric method, which enables an objective description of facial structures, was used to better delineate the craniofacial characteristics of 37 individuals with Prader-Willi syndrome (PWS; 21 males and 16 females; 22 with 15q11q13 deletions and 15 with normal-appearing chromosome 15s) between the ages of 0 to 12 years. Facial parameters were measured from strict frontal and profile photographic 35 mm slides and compared with other facial measurements from the same face (e.g., palpebral fissure width to bizygomatic diameter). We studied 16 photoanthropometric craniofacial indices following the protocols established by Stengel-Rutkowski et al. [1984: Hum Genet 67:272-295] and Butler et al. [1988: Am J Med Genet 30:165-168]. Based on our measurements of 37 Prader-Willi syndrome individuals, none of the parameters were consistently outside of the normal range when compared with photoanthropometric index standards for age established from white control children [Stengel-Rutkowski et al., 1984]. However, several suggestive findings were documented by our analysis including: narrow palpebral fissure width [particularly in older children (6-12 years)], high midface, broad interalar distance, short back of the nose, prominent high chin, and broad low-set ears. No significant differences were found in craniofacial parameters between deletion or nondeletion Prader-Willi syndrome patients with this methodology. These craniofacial parameters (many not previously evaluated in PWS patients) may become useful for early detection, and aid in the diagnosis and the study of the development of the characteristic face seen in Prader-Willi patients.

Isolation of novel cDNA transformation markers from SV40-transformed human keratinocytes.

Differential screening of a cDNA library was used to isolate probes for mRNAs that are induced in simian virus 40 (SV40)-transformed human keratinocytes. Several of these cDNAs hybrid select mRNAs which encode transformation-induced proteins found in the cytoskeletal component of SV40-transformed keratinocytes. One of these cDNAs was used to study the phenotype of normal and transformed cell lines derived from various tissues. We found that mRNA encoding the novel transformation-induced proteins is expressed in two squamous carcinoma cell lines derived from the oral epithelium, four SV40-transformed keratinocyte cell lines, and two SV40-transformed fibroblasts. Normal or transformed lymphoid cells or cell lines derived from carcinoma of the cervix do not express mRNAs which hybridize to these probes. The results from this study suggest that these probes may be used to detect markers of transformation in certain cell types.

A viral-cellular junction fragment from a human papillomavirus type 16-positive tumor is competent in transformation of NIH 3T3 cells.

A 4.4-kilobase DNA fragment (T4.4) from a human tumor (comprising part of the human papillomavirus type 16 E6 promoter; the E6, E7, and part of the E1 open reading frames; and cellular sequences) was found to be competent to fully transform NIH 3T3 cells. This competency resides in the whole hybrid DNA fragment, since the separate viral or cellular DNA sequences were not active. Abundant E6-E7 transcripts were found in the transformed cells. When the cellular fragments were substituted with polyadenylation sequences from polyomavirus or simian virus 40 DNA, little or no restoration of transforming activity was observed. In experiments in which an exogenous reporting gene, that for chloramphenicol acetyltransferase, was used, the possibility was excluded that the cellular flanking sequences act as a traditional enhancer; yet, when the cellular sequences were placed downstream of a chloramphenicol acetyltransferase expression vector (pSV2 CAT), activity of the reference gene was clearly enhanced. These results indicate that DNA containing human papillomavirus type 16 open reading frames E6 and E7 isolated from the genome of a human tumor has transforming potential, that this potential is realized when the viral DNA is joined to cellular sequences, and that the cellular sequences function in a more complex way than by simply providing polyadenylation signals.

[Papilloma viruses in benign and malignant tumors of the mouth and upper respiratory tract]. / Papillomviren in benignen und malignen Tumoren des Mundes und des oberen Respirationstraktes.

A total of 113 benign and malignant tumours of the mouth and upper respiratory tract have been analysed for the presence of papilloma virus DNA. Thirty-four (63%) of 54 papillomatous lesions were found to contain such DNA. Seventy-two percent of the laryngeal papillomas contained HPV 6 or HPV 11 DNA, whereas the oral papillomatoses harboured HPV 6 (15.6%), HPV 11 (9.4%), HPV 7 (9.4%), HPV 13 (12.5%) and HPV 32 (9.4%) DNAs. The high number of HPV 7 DNA-positive lesions was unexpected. In most of the malignant tumours no papilloma-viral DNA was found, with the exception of 3 tongue-base carcinomas, and in a metastasis from one of the tumours, which contained HPV 16 (3 biopsies) and HPV 2 related sequences (1 biopsy).

Infection of the oral mucosa with defined types of human papillomaviruses.

Biopsies from 9 different oral papillomatous proliferations were analysed for human papilloma viral (HPV) sequences of types 1 to 19 and 21 to 26 by Southern blot analysis with 32p-labelled cellular DNA. HPV sequences were detected in 7 out of 9 biopsies obtained from individual patients. Of three cases with the clinical diagnosis focal hyperplasia Heck, two contained HPV-6 related sequences and one HPV 13. In addition, one tongue base papilloma contained HPV 11. A papilloma of the palate revealed HPV 11 sequences. HPV 6 could be demonstrated twice in the remaining 4 oral papillomatous proliferations. Two biopsies remained negative. The data shows that HPV DNA can be regularly demonstrated in oral papillomas.

Mutagenicity studies of a carcinogenic nitrofuran and some analogues.

Earlier studies showed a strong carcinogenicity of the nitrofuran compound 1,2-dihydro-2-(5'nitro-2'furyl)-3-hydroxy-quinazoline-4-one since carcinomas of the urinary bladder in rats and dogs appeared already after six months of treatment. In order to compare possible mutagenic properties of the compound and to get some information on the genotoxic moiety of the molecule, the genotoxicity of four nitrofuran derivatives and of two chemical analogues without the nitrofuran residue was tested in the Ames Test, E. coli WP2 uvrA, and in the rec-assay with Bacillus subtilis. 2-Nitrofuran was also included in this study. Only 2-nitrofuran and the nitrofuran containing derivatives were active in these bacterial test systems. Metabolic activation by liver homogenate was not needed to demonstrate the genotoxic effects. 1,2-dihydro-2-(5'nitro-2'furyl)-3-hydroxy-quinazoline-4-one was more active in the mutagenicity tests in the presence of exogenous metabolic activation. Further studies revealed that this compound was able to induce unscheduled DNA synthesis but was not mutagenic in the micronucleus test.