Normal hepatocytes exhibiting histone H3 with antibody accessible sites that are cryptic in carcinogen-altered hepatocytes.

A Mr 14,000 polypeptide (p14), identified as liver fatty acid binding protein, in normal liver cytosol was shown previously to be the principal target of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene), early during hepatic carcinogenesis in rats. Immunohistochemical analyses using rabbit antiserum against pure p14/liver fatty acid binding protein revealed marked increases in the levels of the protein in cytoplasm specifically during mitosis in normal and regenerating hepatocytes, and throughout the cell cycle in hyperplastic and malignant hepatocytes brought about by carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene) or 3'-methyl-4-dimethylaminoazobenzene. Present also in normal hepatocytes was a nuclear antigen that was not detected in the hyperplastic hepatocytes, benign hepatocytic adenomas, and hepatocellular carcinomas produced by these carcinogens. The nuclear antigen was discerned to be a Mr 17,000 polypeptide (p17) in extracts of normal liver nuclei and nucleosomes. In the present study, the p17 was purified by high-performance liquid chromatography and identified as being the three variants of histone H3, based on common molecular size, amino acid composition, electrophoretic migration in Triton-acetic acid-urea gels, and Western blot and histochemical reactions using affinity-purified antibodies. The histone H3 of all tested organs reacted specifically with the antiserum in Western blots following sodium dodecyl sulfate gel electrophoresis. In contrast, in a survey of 23 normal rat organs, nuclei of virtually only hepatocytes were reactive immunohistochemically. In view of the exceptional immunohistochemical reactivity of nuclei of normal hepatocytes, attributable to accessible histone H3, and the lack of such reaction in carcinogen-altered hepatocytes, the collected evidence indicates that normal hepatocytes contain uniquely available histone H3 sites that become cryptic during the chemical carcinogenesis.

Evidence of functional lymphocytes in some (leaky) scid mice.

Although the majority of severe combined immune deficiency (scid) mice lack functional lymphocytes, some (2-23%) appear to develop a limited number of B and T cells between 3 and 9 mo old. Most of these leaky scid mice were shown to contain very few clones (less than or equal to 3) of Ig-producing plasmacytes. Clonal progeny were distributed unevenly in the lymphatic tissues and appeared as discrete plasmacytic foci. In many cases, individual clones persisted for several months and produced abnormally high concentrations of Ig that included multiple isotypes. Functional T cells were inferred from the ability of leaky mice to reject allogeneic skin grafts, a T cell-dependent reaction. Interestingly, approximately 40% of leaky mice developed thymic lymphomas. In other respects, leaky mice resembled regular scid mice; e.g., their splenic cells failed to express common lymphocyte antigens (Ly-5[B220], Ly-1) and to proliferate in response to lymphocyte mitogens. Histologically, their lymphoid tissues retained the same general pattern of severe lymphocytic deficiency as scid mice.

Iron, ferritin, hepatitis B surface and core antigens in the livers of Chinese patients with hepatocellular carcinoma.

Surgically resected specimens, consisting of tumor and adjacent non-neoplastic liver tissue, were obtained from 40 patients with primary liver cancer at Zhong Shan Hospital, Shanghai Medical University, the People's Republic of China, between March 1983 and July 1984. All were hepatocellular carcinomas (HCC), one being admixed with cholangiocarcinoma. The relationship of hepatitis B virus (HBV) markers with iron and ferritin was evaluated in liver tissues from patients with primary liver cancers. The serum HBsAg (Hepatitis B surface antigen) positive rate was 80.0% (32/40). Cirrhosis was observed in 97.5% (39/40). HBsAg was identified in 82.5% (33/40) of uninvolved liver, and 35.0% (14/40) of HCC tissues (P less than 0.001). HBcAg (hepatitis B core antigen) was detected in 25.0% (10/40) of liver, and 7.5% (3/40) of HCC tissues (P less than 0.05). Stainable iron was found in 65.0% (26/40) of unaffected livers, and 10.0% (4/40) of HCC tissues (P less than 0.001). Ferritin was demonstrated in 75% (30/40) of non-neoplastic liver, and 40% (16/40) of HCC tissues (P less than 0.001). Twenty-two of 33 HCC patients (66.7%) with HBsAg positive cells in their livers also showed stainable iron. Of 16 patients positive for ferritin in HCC cells, iron was found in only two. Iron was found in nine of ten patients with HBcAg in non-neoplastic hepatocytes (P = 0.056); a finding compatible with the hypothesis that iron accumulates in cells replicating HBV. The other results indicate that: immunohistologic ferritin in HCC is not due to increased stainable iron; tumor cells may produce ferritin; polyclonal antibodies to human liver ferritin react better with non-neoplastic hepatocytes than with HCC cells; the high prevalence of HBsAg and cirrhosis in HCC suggests that HBV plays a major etiologic role in hepatocarcinogenesis in China; and one case of HCC is attributed to Schistosoma japonicum infestation via cirrhosis.

Elevated expression and cell cycle deregulation of a mitosis-associated target polypeptide of a carcinogen in hyperplastic and malignant rat hepatocytes.

A cytoplasmic polypeptide with a molecular weight of 14,000 (p14) was previously found to be the principal covalent target protein of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene), early during carcinogenesis in rat liver. The level of immunodetected p14 was markedly increased specifically during all stages of mitosis of hepatocytes in normal and regenerating partially hepatectomized livers. In addition, the polypeptide appeared to be immunologically and behaviorally related to a polypeptide with a molecular weight of 17,500 that is tightly bound to nucleosomes of chromatin in hepatocytes. This report describes the actions of the two polypeptides during hepatocarcinogenesis induced by ingestion of 3'-methyl-4-dimethylaminoazobenzene or N-2-fluorenylacetamide. Both carcinogens acted similarly in bringing about characteristic responses of the two polypeptides, detected immunohistochemically with specific rabbit antiserum and peroxidase-antiperoxidase complex. Four types of discrete hepatocytic lesions were observed. The earliest and least aberrant were hyperplastic foci of proliferating hepatocytes, which generally displayed markedly higher levels of immunostained p14 in cytoplasm, compared to levels in normal diploid hepatocytes. Furthermore, the very high concentrations of p14 were continuously present during cell interphase, in contrast to those in normal and regenerating hepatocytes, in which the elevation is restricted to the period of mitosis. Later-arising lesions were acidophilic adenomas of hepatocytes, which were characterized by quiescent morphology, fairly uniform and bulky eosinophilic cytoplasm, high levels of glycogen, and small delicate nuclei. These cells usually displayed little cytoplasmic p14. Coexistent hepatocytic lesions, i.e., mixed basophilic adenomas, exhibited considerable morphological heterogeneity, often slightly basophilic cytoplasm, and variable immunostain of cytoplasmic p14 during interphase. The fourth lesions were hepatocellular carcinomas, which continuously demonstrated much higher than normal levels of cytoplasmic p14 during cell interphase. During mitosis in the four types of hepatocytic lesions, the levels of immunostained p14 were usually further elevated above those in interphase, regardless of whether they were already very high during interphase in hyperplasia and malignancy, or low in acidophilic adenomas. All four kinds of carcinogen-altered lesions usually displayed little of the detectable Mr 17,500 polypeptide in nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Severe combined immunodeficiency (SCID) in the mouse. Pathology, reconstitution, neoplasms.

Histologic findings in mice with severe combined immunodeficiency (SCID) were remarkably uniform, consisting of lymphopenia, a rudimentary thymic medulla without cortex, relatively empty splenic follicles and lymph nodes, and undeveloped bronchial and gastrointestinal lymphocytic foci. Fluorescence-activated cell sorter studies revealed a few T cells (apparently nonfunctional) in thymus and spleen; interestingly, these cells seemed highly disposed to neoplasia, because thymic T-cell lymphomas were observed in 41 of 269 mice. No pre-B or B cells could be identified. Cells of the myeloid lineage appeared normal. Reconstitution of lymphoid tissues was achieved after intravenous injection of histocompatible bone marrow cells.

Induction of epidermoid differentiation by cyclic adenine nucleotide in cultured mammary tumors of mice.

In search of the degree of responsiveness of mammary adenocarcinomas to signals of differentiation, mouse mammary tumors were induced to undergo a course of development leading to multiple foci of squamous metaplasia, and subsequently a differentiation manifested by marked keratinization. The mammary tumors had spontaneously arisen in the preneoplastic mammary outgrowths of the transplantable lines, D1, MH5, and MH9, after their long-term implantation in gland-free mammary fat pads of virgin BALB/c mice. The inductions were produced in cultured fragments of mammary tumors by incubation for 9 days in the cyclic adenine nucleotide, N6-O2'-dibutyryl cyclic AMP, at 0.1 mM, without or with prostaglandins E1, E2, and B1, each at 5 micrograms/ml, and 1 microM papaverine. The N6-O2'-dibutyryl cyclic AMP alone was as active in the mammary tumors derived from the D1 and MH9 preneoplastic outgrowths as was the entire mixture of inducers. Intracellular cyclic adenine nucleotide may presumably be the specific mediator of the inductive process, presumably being elevated synergistically by entry of the N6-O2'-dibutyryl cyclic AMP, by induction of adenyl cyclase by prostaglandin E1 and E2, and through inhibition of phosphodiesterases by papaverine. Epidermidalization occurred to equal extent in well-differentiated and anaplastic mammary adenocarcinomas, indicative that mammary tumor progression did not affect the susceptibility to this course of development and differentiation. Mammary gland epithelium retains its susceptibility to multifocal epidermidalization in organ culture throughout the gradient of neoplastic transformation and progression toward decreasing growth regulation, starting from normal mammary gland, next preneoplasia (both reported previously), then well-differentiated neoplasia, and lastly anaplastic cancer. The findings support the existence of a common or closely associated pool of progenitor cells for the alveolar and epidermoid courses of development and differentiation in mammary gland. Induction of squamous metaplasia and abundant keratinization in both the well-differentiated and anaplastic mammary adenocarcinomas caused some of the cells to differentiate terminally and to die.

Mitosis in hepatocytes is generally associated with elevated levels of the target polypeptide of a liver carcinogen.

Rat hepatocytes have previously been found to contain in their cytoplasm a 14,000-dalton polypeptide that: is markedly and specifically increased in concentration during the infrequent mitoses that occur in hepatocytes of adult normal liver; is apparently related to a 17,500-dalton polypeptide that is tightly bound to the chromatin of nuclei of normal adult liver; is the principal covalent target of the carcinogen, N-2-fluorenylacetamide (FAA; 2-acetylaminofluorene), early during liver carcinogenesis; is present at highly elevated levels in the proliferating hepatocytes of hyperplastic foci brought about by the two liver carcinogens, FAA and 3'-methyl-4-dimethylaminoazobenzene; and is present at a greatly depressed level in the mitotically nonresponsive parenchyma that surrounds these hyperplastic foci. In the present investigation, we examined the levels of the two polypeptides in hepatocytes undergoing cell division at different rates in livers of diverse normal and regenerative states. Using immunohistochemical techniques, both polypeptides were detected in developing hepatocytes in as early as 15- and 19-day rat fetuses. With the increasing maturity of fetal and neonatal livers in normal rats, a greater percentage of dividing hepatocytes exhibited a higher concentration of the 14,000-dalton target polypeptide than that seen in adjacent interphase hepatocytes. The percentage of mitotic hepatocytes with an elevated level of the polypeptide increased progressively with hepatic development as follows: 38% in 15-day fetuses, 50% in 19-day fetuses, 85% in 1-day neonates, between 79% and 93% until 28 days of age, and finally, 99% in normal adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Target polypeptide of a carcinogen is associated with normal mitosis and carcinogen-induced hyperplasias in adult hepatocytes.

Normal rat liver cytosol was found previously to contain a 14,000-dalton polypeptide that is the principal target of the carcinogen N-2-fluorenylacetamide (2-acetyl-aminofluorene) early during hepatocarcinogenesis. By using antiserum that identifies the 14,000-dalton polypeptide in liver cytosol, an immunologically related 17,500-dalton polypeptide was shown to be present in isolated normal liver nuclei, tightly bound to chromatin. We report here that the 14,000-dalton polypeptide is associated with cell multiplication in normal adult hepatocytes. Of 150 hepatocytes in five stages of mitosis, all invariably displayed a greatly increased concentration of the 14,000-dalton polypeptide in cytoplasm, compared to hepatocytes in interphase, by immunostaining with peroxidase-antiperoxidase complex. In contrast, mitosis did not appear to affect the level of the 17,500-dalton polypeptide in nuclei. A small population of high-polyploid hepatocytes with large nuclei had elevated intensities of cytoplasmic immunostain that approached that in mitotic cells. The increased level of discernible 14,000-dalton polypeptide target of the carcinogen in cytoplasm is a marker of the rare mitotic hepatocytes in normal adult rat liver. Ingestion of the liver carcinogen N-2-fluorenylacetamide for 5 wk or an azocarcinogen, 3'-methyl-4-dimethylaminoazobenzene, for 4 wk brought about early foci of hyperplastic hepatocytes in which there was a great overload of detectable 14,000-dalton target polypeptide in their cytoplasm and a near absence of the 17,500-dalton polypeptide in most nuclei. In contrast, the cytoplasm and nuclei in surrounding morphologically nontransformed hepatocytes of livers of the carcinogen-fed rats immunostained to a much smaller degree than in normal adult hepatocytes. Removal of the azo-dye carcinogen for 4 wk resulted in the disappearance of most hyperplastic foci. A few foci did persist and had similar apparent abnormal levels of the two polypeptides. The high concentration of discernible 14,000-dalton target polypeptide in the cytoplasm of hepatocytes in the hyperplastic foci correlates with their known proliferation, whereas the low cytoplasmic level in the surrounding hepatocytes is consistent with the known inhibition of their mitosis by these carcinogens. The collected evidence appears to joint together a chemical carcinogen, a cytoplasmic principal target polypeptide, a chromatin-bound polypeptide, mitosis in normal adult hepatocytes, early and persistent hyperplastic foci caused by carcinogens, and the inhibition of mitosis by carcinogens in surrounding parenchyma in preneoplastic livers.

Normal liver chromatin contains a firmly bound and larger protein related to the principal cytosolic target polypeptide of a hepatic carcinogen.

A 14,000-dalton polypeptide was previously reported to be the principal protein target of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) in liver cytosol at the start of hepatocarcinogenesis in rats. The 14,000-dalton polypeptide was purified to homogeneity according to gel electrophoreses in both NaDodSO4-containing medium and acetic acid/urea and also by immunogenicity. An immunologically related form of the cytosolic target polypeptide has now been found to be present in the nuclei of normal rat liver as a 17,500-dalton polypeptide that is firmly and ionically bound to chromatin. Serial salt extractions of isolated liver nuclei or chromatin at 0.15 and 0.35 ionic strengths fail to dissolve the bound polypeptide, according to electrophoretic transfer immunoblot analyses. Most of the 17,500-dalton polypeptide is extracted at 0.65 ionic strength, the remainder at 1.2, and none at 2.0, nor thereafter in 8 M urea. In addition, short-term digestion of purified liver nuclei with micrococcal nuclease solubilizes the 17,500-dalton polypeptide. All three protocols also solubilize low levels of intermediate 17,500- to 14,000-dalton species, the latter size being the same as that of the cytosolic protein target of the carcinogen. The presence of protease inhibitors during the isolations and extractions of the nuclei and chromatin reduces the amounts of these smaller polypeptides. In normal rat liver only nuclei and cytoplasm of hepatocytes contain reactive antigen according to peroxidase-antiperoxidase immunohistochemistry, staining most intensely perilobularly, less in the lobular midzone, and least centrilobularly. The nuclei of the perilobular hepatocytes constitute the strongest staining compartment within all of normal liver. Of 22 nonhepatic tissues of normal rats, 16 contain relatively few cells with immunoreactive cytoplasm. Nonhepatic nuclear antigen is present only in villar crest cells of duodenum (which are normally exposed to liver bile), also having cytoplasmic antigen as well. Five kinds of evidence appear to connect the chromatin-bound 17,500-dalton polypeptide of normal liver nuclei to the cytosolic 14,000-dalton polypeptide that is the principal target of the carcinogen early during hepatocarcinogenesis in rats. The present findings indicate a direct connection between a chromosomal protein and the immediate principal cytosolic protein target of a carcinogen.

Persistence of precursor cells of squamous metaplasia in preneoplastic mammary outgrowth lines from mice.

The preneoplastic state is without apparent effect on the induction or prevention of epidermidalization in transplanted mammary outgrowth lines. Development of squamous metaplasia and differentiation (keratinization) were induced in organ cultures of three hyperplastic alveolar and ductular mammary outgrowth lines (D1, MH5, and MH9) that had been extensively passaged in gland-free mammary fat pads of BALB/c virgin mice. The induction was elicited by the mixture of dibutyryl cyclic AMP (0.1 mM), prostaglandins E1, E2, and B1 (each 5 micrograms/ml), and papaverine (1 microM) or by a tenfold higher concentration of dibutyryl cyclic AMP (1 mM) alone for 9 days. The retinoid 2-retinylidene-5,5-dimethyl-1,3-cyclohexanedione at 1 microM and the phorbol ester phorbol 12,13-didecanoate at 10 microM each blocked the induction process. The parameters of the induction and its prevention were analogous in many ways to those previously found with cultures of normal mammary glands of mice and humans, as well as of mouse prostate glands and chick embryo skin. The metaplastic squamous cells that developed in the cultured mammary outgrowths did not proliferate in gland-free mammary fat pads, possibly because the cells were terminally committed or because of insufficient inducers. In contrast, the alveolar and ductular epithelia in the same outgrowths have a transplantable pool of generative cells with the ability to undergo continual proliferation and development. The finding of precursor cells with the potential for epidermoid development and differentiation in the preneoplastic alveolar and ductular outgrowths, despite their extensive serial transplantations, is supportive of the existence of a common or closely associated pool of cells with the ability to develop either into squamous or alveolar mammary epithelium.

Nononcogenic hormone-independent alveoli produced by carcinogens in cultured mouse mammary glands.

Treatment of mouse mammary glands with a high concentration of 7,12-dimethylbenzo(a)anthracene in whole organ culture was reported by Banerjee et al. to transform foci of lobuloalveoli to a hormone-independent state, and to give rise to mammary hyperplastic outgrowths and adenocarcinomas in vivo. In the present study using the identical system, mammary glands of BALB/c mice were exposed to 7,12-dimethylbenzo(a)anthracene or N-2-fluorenylacetamide at low concentrations that bring about maximal incidences of the hormone-independent hyperplastic lobuloalveolar lesions with minimal cytotoxicity. After morphological development of the lobuloalveoli in culture, the glands were enzymatically dissociated into cells and inoculated into gland-free inguinal mammary fat pads of syngeneic mice bearing pituitary gland implants during the initial 8 weeks. After 11 months, fragments of the resultant mammary outgrowths from each mouse were implanted into the gland-free inguinal mammary fat pads of 3 syngeneic mice (not bearing pituitary gland supplements) and were permitted to grow for another 11 months. Mammary outgrowths from the primary and secondary implants were neither neoplastic, anaplastic, nor dysplastic. Also, no hyperplasia in any mammary outgrowth could be attributed to the action of either carcinogen, especially when outgrowths were compared with contralateral outgrowths that arose from the control glands exposed to dimethyl sulfoxide (solvent of the carcinogens) in culture and/or with untreated thoracic mammary glands of the same hosts. One interpretation of these findings is that the hormone-independent, hyperplastic alveolar lesions may not be an appropriate in vitro marker of oncogenic transformation by chemical carcinogens in culture. The great variety of procarcinogens and activated carcinogens that bring about this lesion in vitro and its morphological similarity to presumptive mammary preneoplastic lesions in vivo weigh against this interpretation. A second hypothesis is that high concentrations of procarcinogens, despite their considerable cytotoxicity, complete a multistep process of oncogenic transformation in surviving mammary epithelium, whereas low concentrations optimized to produce the lesions in maximal number do not.

A severe combined immunodeficiency mutation in the mouse.

The most debilitating human lymphoid deficiency disease, known as severe combined immunodeficiency (SCID), impairs the differentiation of both T and B lymphocytes. Affected infants are highly susceptible to recurring infections of viruses, fungi and bacteria and invariably die within 2 yr of birth. Inheritance of this congenital syndrome may show X-linked or autosomal recessive control. To date autosomal recessive inheritance of SCID has been observed in Arabian foals which represent the only known animal model of this disease syndrome but here we report an autosomal recessive mutation in mice that severely impairs lymphopoiesis. Mice homozygous for this mutation have few if any lymphocytes; consequently they are hypogammaglobulinaemic and deficient for immune functions mediated by T and B lymphocytes. These mice, therefore, represent a new model for investigating how lymphoid differentiation may be impaired in the disease state and regulated in the normal state.

Induction of squamous metaplasia: requirement for cell multiplication, and competition with lobuloalveolar development in cultured mammary glands.

Mouse mammary glands were previously shown to undergo either of two courses of development and differentiation in whole organ culture. The combination of insulin, prolactin, aldosterone, and hydrocortisone induces a structural development of lobuloalveoli, followed by casein production. In the second course, the mixture of dibutyryl cyclic AMP, prostaglandins E1, E2 and B1, and papaverine brings about an extensive squamous metaplasia and excessive keratinization. In the present study, the foci of the metaplastic squamous cells appeared to originate from single or very few cells. A preferential stimulation of squamous cell multiplication was involved in the induction process. Twice the relative number of nuclei incorporated 3H-thymidine in the squamous metaplastic cells than in the surrounding cuboidal epithelium, according to autoradiography. The necessity for cell multiplication was indicated by the reversible and complete inhibitions of both the metaplastic squamous development and 3H-thymidine incorporation by 1 mM hydroxyurea in the culture medium. Simultaneous inductions of both courses of development and differentiation revealed a competitive and reciprocal relationship between the two pathways. The concurrent expressions of both courses were considerably less than those achieved when either pathway was induced alone. Only the combination of the three types of inducers of squamous metaplasia was able to compete effectively with the hormonal induction of lobuloalveolar development and differentiation. The findings suggest that individual metaplastic squamous foci may originate as clones of cells by processes that require cell multiplication, rather than through a direct non- replicative conversion of pre-existent cells of the cuboidal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Totipotent hematopoietic stem cells: normal self-renewal and differentiation after transplantation between mouse fetuses.

Successful engraftment of mouse fetal liver cells in early fetal recipients, after microinjection via the placental circulation, is attributable to seeding of the recipient's liver by a cell type that is ancestral to both the myeloid and lymphoid definitive lineages and is capable of sustained self-renewal and differentiation for more than 2 years. This primitive cell is therefore the normal totipotent hematopoietic stem cell (THSC). The use of a large series of mutant anemic recipients with decreasing severity of an endogenous stem-cell defect (W/W, Wv/Wv, Wf/Wf, Wv/+), and therefore of graded selective advantage to normal donor cells, has revealed that engraftment entails marginal numbers of cells--probably individual ones--in the least afflicted hosts. Thus the observed progressive and coordinate shift toward donor-strain erythrocytes, granulocytes and B and T lymphocytes, over time, indicates THSC expansion to form a larger stem-cell pool and normally regulated differentiation of cells from the pool. This transplant system allows allogeneic combinations with impunity and therefore provides many novel experimental possibilities for investigating THSC normal development, genetic abnormalities or neoplastic potential in relation to the intact developmental succession of hematopoietic tissue environments in vivo.

Enhancement of squamous cell development in cultured skin by cyclic adenine nucleotide and prostaglandins.

The present study tests the hypothesis that agents known to elevate the level of intracellular cyclic adenine nucleotide may direct different epithelial cells onto a pathway of epidermoid (squamous) development and differentiation. We report here that the mixture of dibutyryl cyclic AMP (dbcAMP), prostaglandins E1, E2 and B1 (PG E1, E2, B1), and papaverine (pap) enhances the rate of normal squamous cell development in organ-cultured skin of chick embryos. The three components may act synergistically to elevate the level of intracellular cyclic adenine nucleotide. We recently reported that the same group of agents induces abnormal development (squamous metaplasia) and aberrant differentiation (keratin production) in the normally cuboidal epithelium of cultured whole mammary glands of mice [1]. Thus, dbcAMP, PG E1, E2, B1, and pap are effective in enhancing normal squamous cell development and also in inducing squamous metaplasia de novo in the epithelial components of two different organs of embryonic and adult animals of two classes of vertebrates. The combined findings are suggestive that cyclic adenine nucleotide together with the prostaglandins may act generally on diverse types of epithelia to bring about squamous cell development and a differentiation marked by keratin production.